BIOLOGY 

LIBRARY 

G 


ESSENTIALS 

OF 

PATHOLOGICAL  CHEMISTRY 


Including  Description  of  the  Chemical  Methods 
Employed  in  Medical  Diagnosis 


BY 


VICTOR  C.MYERS,  M.  A.,  PhD.,  and  MORRIS  S.  FINE,  Ph.D. 

Professor  of  Pathological  Chemistry,        Instructor  in  Pathological  Chemistry, 

Xew  York  Post-Graduate  Medical  New  York  Post-Graduate  Medical 

School  and  Hospital  School  and  Hospital 


Reprinted  from 

The  Post-Graduate,  1912-13 

New  York 

1913 


KB  to 
Ml 


BIOLOGY 
LIBRARY 

a 


Copyright  1913 
By  V.  C.  MYERS  and  M.  S.  FINE 


i- 


PREFACE 

The  individual  chapters  of  this  volume  are  reprinted  from 
the  Post-Graduate,  in  which  they  have  appeared  at  intervals 
during  the  past  year.  They  have  been  designed  primarily  as  a 
guide  for  the  classes  in  Elementary  Pathological  Chemistry  in 
this  laboratory,  although  the  tests  described  are  likewise  em- 
ployed in  the  examination  of  the  routine  hospital  specimens. 

The  various  chapters  have  been  written  with  the  idea  that  an 
adequate  appreciation  of  normal  physiological  functions,  is  a 
necessary  prerequisite  to  an  intelligent  conception  of  pathological 
chemical  processes,  especially  since  the  latter  may  be  regarded  as 
deviations  from  the  normal.  It  will  be  observed  that  many  of 
our  diagnostic  tests  have  for  their  purpose  the  detection  of  an 
inability  on  the  part  of  the  body  to  complete  or  properly  carry  out 
some  of  the  normal  physiological  transformations.  In  harmony 
with  this  view,  a  consideration  of  the  physiology  of  digestion  in 
Chapters  I  and  II  is  followed  by  the  abnormal  variations  fre- 
quently encountered,  and  methods  by  which  such  abnormalities 
may  be  detected.  Likewise  Chapters  V  and  VI  deal  with  the 
normal  mechanism  of  carbohydrate  and  fat  metabolism,  before 
indicating  how  certain  functional  defects  may  lead  to  glucosuria 
and  acetonuria. . .  As  will  be  observed,  the  general  arrangement 
of  the  chapters  follows  the  course  of  the  various  food  materials 
through  the  body. 

Each  chapter  is  composed  of  a  resume  of  the  subject  with  which 
it  deals,  followed  by  directions  for  laboratory  tests,  especially 
those  which  are  considered  of  diagnostic  value.  The  authors  have 
endeavored  to  make  the  discussions  complete,  though  concise, 
presenting  the  subject  with  the  most  recent  developments  in 
mind.  The  "  Laboratory  Procedures  "  contain  descriptions  of 
tests,  which  for  the  most  part  are  very  simple  and  require  only 
a  limited  equipment.  However,  tests  and  determinations  are 
occasionally  included  which  are  not  especially  simple  and  neces- 
sitate considerable  manipulation,  but  which,  nevertheless, 
frequently  yield  valuable  data.     This  is  true  of  the  methods  for 


989763 


iv.  PREFACE. 

blood  analysis  described  in  Chapter  X.  In  the  instances  where 
several  tests  have  been  described,  preference  is  indicated  by  the 
order  of  description. 

In  the  preparation  of  this  volume  the  authors  have  been  guided 
primarily  by  the  recent  literature  upon  the  subjects  discussed, 
though  frequent  reference  has  been  made  to  many  of  the  stand- 
ard texts  on  this  and  allied  subjects,  a  list  of  which  has  been 
given  on  page  130. 

The  authors  take  this  opportunity  of  acknowledging  the  kind 
cooperation  of  Dr.  Jonathan  Wright,  Director  of  the  Department 
of  the  Laboratories,  not  only  in  the  preparation  of  this  guide  for 
their  classes  but  also  in  the  various  other  undertakings  of  the  lab- 
oratory. They  also  desire  to  express  their  appreciation  to  the 
Editors  of  the  Post-Graduate,  Drs.  C.  G.  Heyd  and  O.  S.  Hillman, 
for  their  kindness  and  advice  in  the  publication  of  the  different 
chapters,  and  to  Dr.  Ludwig  Kast  for  valuable  suggestions.  The 
drawings  and  microphotographs  of  urinary  sediments  and  photo- 
graphs of  apparatus  were  made  for  us  by  Mr.  K.  K.  Bosse.  For 
the  reproduction  of  Fig.  14,  we  are  indebted  to  the  New  York 
Medical    Journal. 

V.  C.  M. 
M.  S.  F. 
New  York, 
July,  1913. 


CONTENTS 

CHAPTER  PAGE 

I .  Gastric     Digestion,     Including  .  Methods     of 

Gastric  Analysis 1 

II .  Digestive  Changes  in  the  Intestine  Together 

with  the  Formation,  Composition  and  Clinical 

Significance  of  the  Feces. 12 

III .  The      Physical      Properties,      Inorganic      and 

Organic  Physiological  Constituents  of  Urine  28 

IV.  Albuminuria. 49 

V.  Glucosuria  and  Other  Types  of  Mellituria  .  ...  60 

VI .  Acidosis 79 

VII  .       PlGMENTURIA 88 

VIII.  Examination  of  Urinary  Sediments 96 

IX .  The  Chemistry  and  Physiology  of  Milk 103 

X .  Blood  and  Other  Body  Fluids , 114 

Appendix.     Laboratory  Suggestions 125 

Index 131 


Digitized  by  the  Internet  Archive 

in  2008  with  funding  from 

Microsoft  Corporation 


http://www.archive.org/details/essentialsofpathOOmyerrich 


ESSENTIALS  OF 
PATHOLOGICAL    CHEMISTRY. 


CHAPTER   I. 
Gastric  Digestion  Including  Methods  of  Gastric  Analysis. 

Man  ingests  daily  varying  amounts  of  protein,  fat  and 
carbohydrate,  together  with  water  and  inorganic  salts.  The 
quantities  of  these  constituents  depend  upon  the  age,  habits  and 
occupation  of  the  individual;  sufficient  food,  primarily  carbo- 
hydrate and  fat,  being  necessary  to  supply  the  body  with  the 
required  energy  for  heat  and  muscular  work.  This  amounts  to 
about  40  calories  per  kilogram  of  body  weight,  including  suffi- 
cient protein  to  build  up  the  tissues  and  to  make  good  the  wear  and 
tear  of  the  body.  Just  the  quantity  of  this  protein  food  essen- 
tial for  our  daily  needs  is  not  quite  clear  at  the  present  time.  The 
dietaries  of  Voit  and  of  Atwater  call  for  upwards  of  125  grams  of 
protein,  but  Chittenden  has  recently  pointed  out  that  the  body 
can  be  maintained  in  perfect  health  on  half  this  amount. 

Whatever  this  food  intake  may  be,  to  render  service  to  the 
body,  it  must  undergo  numerous  changes,  both  before  and  after 
its  absorption.  The  first  step  in  the  digestion  of  the  food, 
aside  from  the  mechanical  factor  of  mastication,  is  the  action  of 
the  ptyalin  of  the  saliva  upon  the  starchy  foods  with  the  pro- 
duction of  dextrins  and  maltose,  this  action  being  long  continued 
in  the  fundic  portion  of  the  stomach.  With  the  taking  of  food 
into  the  mouth,  normally  a  psychic  secretion  of  gastric  juice  is 
started,  and  with  the  taking  of  certain  substances  into  the  stom- 
ach, e.g.,  meat  extracts,  soup,  etc.,  the  secretion  is  augmented, 
and  again  still  further  increased  by  certain  of  the  products  of 
digestion.  The  essential  constituents  of  this  secretion  are  the 
hydrochloric  acid  (in  about  four-tenths  per  cent,  concentration) 
and  the  enzymes,  pepsin  and  rennin.  The  curdling  action  of 
rennin  on  milk  is  well  known,  though  just  its  function  in  digestion 

is  not  so  clear. 

1 


2  PATHOLOGICAL  CHEMISTRY 

The  pepsin  J  secreted  in  the  inactive  form,  pepsinogen,  and 
activated  by.  the  hydrochloric  acid,  produces  important  pre- 
liminary transformations  in  the  protein.  After  the  latter  has 
been  acted  upon  by  the  HC1  and  caused  to  swell,  hydrolytic 
cleavage  of  the  protein  is  begun,  and  normally  before  leaving  the 
stomach,  it  has  been  broken  down  into  proteoses  and  peptones. 
It  will  be  well  to  bear  in  mind  that  gastric  digestion  together 
with  pancreatic  and  intestinal  digestion  has  much  more  to  do 
than  to  render  the  food  material  soluble.  Without  exception, 
the  protein,  fat  or  carbohydrate  is  broken  down  into  the  simplest 
cleavage  products,  which  are  then  resynthesized  to  form  the  body 
protein,  body  fat  or  glycogen. 

Another  factor  of  great  importance  is  the  mechanical  one, 
upon  which  the  X-ray  observations  of  Cannon1  have  recently 
thrown  much  light.  The  stomach  is  always  only  so  large  as  its 
contents,  the  function  of  the  fundic  portion  being  mainly  that 
of  a  reservoir.  A  few  minutes  after  the  entrance  of  food  into 
the  stomach,  peristaltic  waves  start  in  the  prepyloric  portion 
and  run  towards  the  pylorus.  When  a  sufficient  concentration 
of  the  hydrochloric  acid  has  been  reached,  the  pyloric  sphincter 
relaxes  and  the  advancing  constrictions  squeeze  some  of  the  mate- 
rial into  the  duodenum.  The  presence  of  the  hydrochloric  acid 
in  the  duodenum  again  causes  the  pyloric  sphincter  to  close,  and 
it  is  obvious  that  the  waves  running  to  the  closed  pylorus  serve 
to  thoroughly  mix  the  food  with  the  digestive  juice.  It  is  worthy 
of  note  that  secretion  of  the  gastric  juice  takes  place  in  this 
region  over  which  the  peristaltic  waves  pass.  Of  the  foods  pas- 
sing the  pylorus,  the  protein  should  normally  be  found  largely  in 
the  proteose-peptone  stage,  and  the  starchy  material  in  the  dex- 
trin stage,  but  the  fat  will  be  found  essentially  unaltered.  Prac- 
tically no  absorption  takes  place  in  the  stomach  under  ordinary 
conditions. 

During  the  course  of  24  hours,  a  very  large  amount  of  gastric 
juice  is  secreted,  probably  over  two  liters.  However,  in  the  non- 
digesting  stomach,  only  a  very  small  amount  of  fluid,  one  to  sixty 
cc.  is  found  to  be  present  under  normal  conditions,  though  patho- 
logically ^large  amounts  are  sometimes  observed.  The  clinical 
examination  of  the  gastric  juice  is  generally  made  after  the  secre- 

1.  Cf.  Cannon:  The  Mechanical  Factors  of  Digestion,  London  and 
New  York,  1911. 


GASTRIC  DIGESTION  3 

tion  has  been  excited  to  activity  by  some  definite  test  meal  for 
a  definite  length  of  time.  The  test  meal  of  Ewald-Boas,  de- 
scribed below,  is  the  one  quite  universally  employed.  The 
normal  gastric  contents  as  here  obtained  with  the  stomach 
tube  is  a  white  or  light  brown  fluid  with  finely  divided  or  pulpy 
bread  in  the  sediment.  The  odor  is  not  strongly  sour,  and  the 
amount  of  mucus  is  scanty. 

Volume. — The  quantity  of  fluid  obtained  at  the  end  of  an 
hour  after  the  Ewald  test  meal  may  normally  vary  between  50 
and  100  cc.  Larger  amounts,  200  to  300  cc,  are  indicative  of  di- 
minished motility  or  hypersecretion,  while  very  large  amounts, 
500  to  4000  cc,  suggest  dilatation  of  the  stomach,  and  usually 
benign  or  malignant  stenosis  of  the  pylorus. 

Acidity. — The  amount  of  free  HC1  found  by  the  Topfer 
method  (see  below)  after  a  test  meal  in  normal  individuals 
averages  between  30  and  40,  but  as  high  as  80  or  90  may  be  ob- 
served in  certain  pathological  conditions,  while  in  others,  HC1 
may  be  entirely  absent.  The  secretion  of  HC1  is  decreased  in 
a  variety  of  chronic  diseases,  including  carcinoma  of  the  stomach 
and  advanced  chronic  gastritis;  while  in  neurasthenic  and 
hysterical  individuals,  it  may  be  entirely  absent.  On  the 
other  hand,  an  hyperchlorhydria  may  be  met  with  in  a  beginning 
gastritis,  in  continuous  hypersecretion  and  in  certain  cases  of 
gastric  ulcer. 

The  total  acidity  usually  lies  between  50  and  80,  and  is  de- 
pendent upon  somewhat  the  same  factors  as  the  free  HC1.  The 
combined  HC1  averages  10  to  15,  and  together  with  the  free  HC1, 
represents  the  useful  acid  secretion. 

The  absence  of  the  HC1  affords  rather  favorable  oppor- 
tunity for  bacterial  action  in  the  stomach,  especially  of  a 
fermentative  nature.  Lactic  acid  is  a  very  common  product  of 
this  bacterial  fermentation.  Its  presence  in  large  amounts  is 
suggestive,  though  not  pathognomonic,  of  carcinoma.  Quite 
generally,  this  large  amount  of  lactic  acid  is  accompanied  by 
the  presence  of  the  Boas-Oppler  bacillus,  which  probably  plays 
an  important  part  in  the  lactic  fermentation.  This  organism, 
probably  identical  with  the  Bulgarian  bacillus,  is  found  in 
75  to  85  per  cent,  of  the  cases  of  carcinoma  of  the  stomach 
but  seldom  in  other  conditions.  Volatile  fatty  acids,  chiefly 
butyric  and  acetic,  sometimes   accompany    fermentative   con- 


4  PATHOLOGICAL  CHEMISTRY 

ditions  of  the  stomach,  but  are  of  much  less  clinical  significance 
than  the  lactic  acid. 

Enzymes. — The  absence  of  the  gastric  enzymes,  pepsin  and 
rennin,  is  distinctly  less  common  than  that  of  HC1.  In  prac- 
tically all  cases  in  which  sufficient  HC1  is  present,  there  is 
an  abundance  of  pepsin.  The  estimation  of  pepsin  is  chiefly  of 
value  in  cases  suggesting  an  advanced  lesion  of  the  gastric  mucosa, 
and  in  which  free  HC1  has  been  shown  to  be  absent.  The 
results  of  Rose1  indicate  that  this  is  an  important  diagnostic  test 
in  carcinoma  of  the  stomach. 

Blood. — Hemorrhage  from  the  stomach  may  be  observed  in 
the  most  diverse  conditions.  It  may  have  a  primary  origin 
as  in  ulcer  and  carcinoma,  or  appear  secondarily  to  diseases 
of  other  organs,  leading  to  a  hyperemic  condition  of  the  gastric 
mucosa. 

Motility. — Considerable  information,  as  stated  above,  may 
be  obtained  with  regard  to  the  motility  of  the  stomach  from 
the  Ewald  meal.  If  the  activity  is  excessive,  little  or  no  gastric 
contents  will  be  obtained;  while  if  the  motor  activity  is  dimin- 
ished or  the  organ  is  dilated,  a  large  quantity  of  fluid  will  be 
obtained.  To  secure  more  accurate  data  in  this  regard,  it  is 
quite  customary  to  resort  to  some  retention  meal,  and  in  that 
way  obtain  a  more  accurate  idea  with  regard  to  the  retention  of 
food  in  the  stomach.  In  place  of  the  regular  evening  meal,  the 
patient  may  be  given  a  plate  of  porridge,  cooked  with  rice  or 
raisins,  and  one  or  two  slices  of  bread  and  butter,  or  as  is  employed 
in  this  hospital,  four  ounces  each  of  boiled  string  beans  and  rice.2 
At  seven  or  eight  a.m.  the  next  morning,  the  stomach  is  aspirated 
and  the  return  examined  for  evidences  of  retention.  Normally 
there  should  be  no  retention  of  food.  The  acidity  will  be  found 
to  correspond  quite  well  with  that  of  the  Ewald  meal,  which  is 
conveniently  given  subsequently  to  the  removal  of  this  retention 
meal. 

Test-Meal. — The  test-breakfast  of  Ewald  and  Boas  is  used 
almost  exclusively  in  the  examination  of  gastric  juice.  This 
consists  of  two  slices  of  bread  without  butter,  a  glass  of  water, 


1.  Rose:     Arch.  Int.  Med.,  1910,  V.,  p.  459. 

2.  Many  practical  suggestions  with  regard  to  various  laboratory  tests 
have  recently  been  given  by  Coffen:  Post-Graduate,  1911,  XXVI, 
p.  274. 


GASTRIC  DIGESTION  5 

and  a  cup  of  tea  without  milk  or  sugar;  or,  more  accurately, 
about  50  grams  of  bread  and  400  cc.  of  fluid.  The  test-breakfast 
should  be  eaten  by  the  patient  on  an  empty  stomach,  allowing 
ten  minutes  for  its  consumption,  and  exactly  one  hour  later, 
should  be  siphoned  from  the  stomach  by  an  ordinary  soft  rubber 
tube. 

LABORATORY    PROCEDURES. 

Before  filtering  the  contents  of  the  stomach,  the  general 
physical  characteristics  named  below  should  be  noted  and 
recorded : 

1.  Color. — Recorded  with  the  idea  of  possible  detection  of 
b1ood  or  bile. 

2.  Consistency. — Noted  with  special  reference  to  possible 
presence  of  increased  amounts  of  mucus. 

3.  Odor. — Whether  normal  (faintly  sour)  or  fetid  (rancid). 

4.  Mucus. — Detected  by  the  consistency. 

5.  Sediment. 

a.  Quantity. — Whether  the  meal  has  been  well  or  poorly 
digested.  If  well  digested,  there  should  be  a  layer  of  finely- 
divided  bread  residue  on  the  bottom  of  the  glass  containing 
the  stomach  contents,  and  over  this  should  be  a  layer  of 
semi-transparent  gastric  juice;  if  poorly  digested,  the 
stomach  contents  will  consist  of  Only  a  small  quantity  of 
fluid  and  many  coarse  lumps  of  bread. 

b.  Character. — Whether  blood,  pus  or  stagnant  remnants  of 
food  are  mixed  with  the  test-breakfast. 

6.  Volume. — The  total  volume  is  measured  in  cc.  and  then 
filtered  through  a  folded  filter  preparatory  to  the  chemical 
examination. 

7.  Detection  of  Free  Hydrochloric  Acid. — The  presence  of  free 
HCl  is  very  readily  detected  with  a  drop  of  congo  red1  or  of 
Topfer's2  reagent,  the  congo  red  being  turned  blue  and  Topfer's 
reagent  a  bright  cherry  red  in  the  presence  of  this  acid.  For 
qualitative  tests  the  reagents  are  conveniently  used  in  the  form 
of  test  papers. 

8.  Quantitative  Determination  of  the  Acidity. — If  the  free  acid- 
ity,  HCl,  and    total  acidity  are  the  only  results  desired,  these 

1.  One-half  gram  of  congo  red  dissolved  in  90  cc.  of  water  and  10  cc. 
of  95  per  cent,  alcohol  added. 

2.  One-half  gram  of  dimethylaminoazobenzene  dissolved  in  100  cc.  of 
95  per  cent,  alcohol. 


6  PATHOLOGICAL  CHEMISTRY* 

figures  can  very  easily  be  determined  in  the  same  solution. 
Ten  cc.  (or  five  cc.  if  necessary)  of  the  filtered  gastric  contents  are 
introduced  into  a  porcelain  dish  and  the  free  acidity  titrated 
with  N/10  NaOH1,  using  three  drops  of  Topfer's  reagent  as 
indicator.  When  the  initial  pinkish-red  color  has  been  replaced 
by  a  bright  yellow  color,  the  reading  is  taken,  and  three  drops 
of  a  one  per  cent,  alcoholic  solution  of  phenolphthalein2  added. 
The  solution  is  titrated  until  a  distinct  pink  color  reappears  (total 
acidity) . 

Topfer's  Method. — Occasionally  it  may  be  desirable  to  deter- 
mine the  combined  acidity  and  acidity  due  to  organic  acids  and  acid 
salts,  in  addition  to  the  total  and  free  acidity.  Three  10  cc. 
portions  are  titrated  with  N/10  NaOH,  using  for  indicators: 

1.  The  physician  will  find  it  most  convenient  to  secure  the  alkali  of 
proper  strength  already  prepared,  but  where  considerable  quantities  are 
needed,  sodium  hydroxide  of  N/10  strength  may  be  accurately  prepared 
by  a  simple  formaldehyde  titration  method  from  N/10  ammonium  sul- 
phate. Ammonium  sulphate  is  an  anhydrous  salt  and  can  be  obtained 
perfectly  pure. 

a.  3.304  gms.  of  ammonium  sulphate  (one-fortieth  of  the  molecular 
weight)  are  dissolved  in  distilled  water  and  made  up  to  exactly  500  cc. 
in  a  volumetric  flask. 

b.  A  few  drops  of  a  one  per  cent,  alcoholic  solution  of  phenolphthalein 
are  added  to  50-100  cc.  of  commercial  formalin  and  dilute  sodium  hy- 
droxide added  until  any  formic  acid  present  is  neutralized,  indicated  by 
the  appearance  of  the  first  permanent  faint  pink  color. 

c.  About  4  grams  of  sodium  hydroxide  are  dissolved  in  about  800  cc. 
distilled  water,  and  a  portion  placed  in  a  burette. 

d.  Exartly  10  cc.  of  the  N/10  ammonium  sulphate  are  now  pipetted 
into  a  beaker,  five  cc.  of  the  neutralized  formalin  added  and  the  solution  tit- 
rated with  the  sodium  hydroxide  to  the  first  permanent  pink.  This  pro- 
cess is  repeated  5  to  10  times  until  one  is  sure  of  the  exact  strength  of  the 
hydroxide,  and  the  required  amount  of  water  added  to  the  remainder  of  the 
800  cc.  to  dilute  to  decinormal  strength. 

When  a  neutral  solution  of  an  ammonium  salt  is  treated  with  formalde- 
hyde, combination  occurs  with  the  formation  of  hexamethylenetetramine 
(urotropin)  and  the  liberation  of  a  corresponding  amount  of  titratable  acid. 

From  the  N/10  alkali,  N/10  hydrochloric  acid  may  be  prepared. 

A  series  of  gravimetric  determinations,  which  we  have  made  upon  N/10 
sulphuric  acid  standardized  to  N/10  sodium  hydroxide  prepared  in  this 
way,  has  shown  that  this  method  is  sufficiently  accurate.  For  example, 
25  cc.  of  N/ 10  sulphuric  acid  should  give  0.2918  gm.  of  barium  sulphate. 
Eleven  determinations  resulted  as  follows:  0.2928,  0.2918,  0.2922, 
0.2912,  0.2924,  0.2920,  0.2928,  0.2920,  0.2914,  0.2926,  0.2918  gm.  giving 
an  average  of  0.2921  gm.,  an  error  of  one-tenth  of  one  per  cent. 

2.  One  gram  of  phenolphthalein  dissolved  in  100  cc.  of  95  per  cent,  alcohol 


GASTRIC  ANALYSIS  7 

a.  Topfer's  reagent  (free  acidity), 

b.  Alizarin1  (combined  acidity), 

c.  Phenolphthalein  (total  acidity). 

In  the  case  of  the  alizarin  titration,  the  red  color,  which  appears 
after  the  tinge  of  yellow  (due  to  the  addition  of  the  indicator)  has 
disappeared,  must  be  replaced  by  a  distinct  violet  color.  In- 
asmuch as  the  alizarin  reacts  with  all  but  the  combined  acidity, 
this  acidity  is  obtained  by  subtracting  the  alizarin  result  from 
that  of  the  phenolphthalein. 

The  various  acidities  are  generally  expressed  by  the  number 
of  cc.  of  N/10  NaOH  necessary  to  neutralize  100  cc.  of  gastric 
juice. 

9.  Lactic  Acid. — In  the  absence  of  free  HC1,  fermentation 
may  take  place  in  the  stomach  with  the  formation  of  lactic  acid. 

a.  Kelling  Method. — To  a  test-tubeful  of  water,  a  drop  or 
two  of  10  per  cent,  ferric  chloride  is  added,  so  that  the 
liquid  is  barely  colored.  One-half  is  then  poured  into  a  second 
tube  and  serves  as  a  control.  A  small  amount  of  the  gastric  fil- 
trate is  added  to  the  other  specimen,  when  in  the  presence  of  lactic 
acid,  a  distinct  yellow  develops  at  once,  which  appears  the  more 
marked  when  compared  with  the  nearly  colorless  control. 

b.  Strauss  Method. — To  detect  lactic  acid,  it  is  always  pre- 
ferable to  remove  it  from  disturbing  factors,  and  the  Strauss 
method  also  permits  of  a  rough  estimation  of  the  amount  of 
lactic  acid  present.  Five  cc.  of  gastric  contents  are  placed  in 
a  small  graduated  separatory  funnel,  20  cc.  of  ether  added, 
and  the  mixture  thoroughly  shaken.  As  soon  as  the  ether 
has  separated,  the  solution  is  allowed  to  run  out,  except  the 
uppermost  five  cc.  of  ether.  Twenty  cc.  of  water  and  two 
drops  of  10  per  cent,  ferric  chloride  are  added  and  the  whole 
gently  shaken.  One-tenth  per  cent,  of  lactic  acid  gives  a  very 
intense  yellowish-green  color;  fi^e-hundredths  per  cent,  a  slight 
green  color. 

10.  Test  for  Pepsin. — Pepsin  may  be  detected  quite  simply  as 
follows:  The  acidity  of  10  cc.  of  filtered  gastric  contents  is 
brought  up  to  0.2  to  0.5  per  cent,  with  HC1  and  a  flock  of  fibrin 
or  bit  of  coagulated  egg  white  added  to  this  tube  and  a  control  of 
two-tenths  per  cent.  HC1.     The  tubes  are  placed  in  the  incubator 


1.   One  gram  of  sodium  alizarin  sulphonate  dissolved  in  100  cc.  of  water. 


8  PATHOLOGICAL  CHEMISTRY 

for  one  to  two  hours  and  any  digestion  observed.     The  quantita- 
tive test  below  is  nearly  as  simple  and  much  more  satisfactory. 
11.  Estimation  of  Pepsin. — The  method  of  Rose1  is  both  con- 
venient and  accurate.     The  solutions  required  are: 

a.  0.25  gm.  of  pea  globulin  dissolved  in  100  cc.  of  10  per 
cent,  sodium  chloride  solution.2 

b.  0.6  per  cent,  hydrochloric  acid  solution.3 

c.  A  measured  volume  of  stomach  contents  neutralized 
to  litmus  paper  with  dilute  alkali  and  diluted  to  five  times 
the  original  volume  with  water;  the  gastric  contents  thus 
diluted  is  divided  into  two  parts,  one  of  which  is  boiled. 

The  determination  is  carried  out  as  follows:  In  each  of  a 
series  of  six  test  tubes  is  placed  one  cc.  of  the  globulin  solution  and 
one  cc.  of  the  acid.  The  unboiled  gastric  juice  is  then  added  in 
increasing  amounts,  with  the  final  addition  of  the  boiled  juice 
to  render  equal  the  volume  of  fluid  in  each  tube.  The  following 
scheme  will  illustrate  this  arrangement : 

Tubes  12         3         4         5         6 

Globulin  solution cc.  1.0  1.0  1.0  1.0  1.0  1.0 

Hydrochloric  acid cc.  1.0  1.0  1.0  1.0  1.0  1.0 

Unboiled  gastric  juice.  . .  cc.                 0  0.1  0.3  0.5  0.8  1.0 

Boiled  gastric  juice cc.  1.0  0.9  0.7  0.5  0.2  0 

Total  volume cc.  3.0     3.0     3.0     3.0     3.0     3.0 

Total  acidity per  cent  HC1     0.2     0.2     0.2     0.2     0.2     0.2 

The  tubes  are  then  shaken  and  incubated  at  50  to  52°  C.  for  15 
minutes  or  at  35  to  36°  C.  for  one  hour.  The  enzyme  content  is 
expressed  by  the  number  of  cc.  of  the  globulin  solution  that  would  be 
digested  by  one  cc.  of  the  undiluted  gastric  juice  under  the  above  con- 
ditions.   Normally  an  enzyme  content  of  8  to  11  may  be  obtained. 

1.  Rose:      Arch.  Inst.  Med.,  1910,  V,  p.  459. 

2.  Pea  globulin  may  be  prepared  by  grinding  up  a  couple  of  handfuls 
of  garden  peas,  extracting  with  about  200  cc.  of  10  percent,  sodium  chloride 
filtering  and  then  pouring  the  filtrate  into  about  a  liter  of  distilled  water. 
The  insoluble  pea  globulin  settles  to  the  bottom  and  is  filtered  off  and 
dried  at  a  low  temperature.  A  0.25  per  cent,  solution  of  this  is  now  pre- 
pared in  10  per  cent,  sodium  chloride. 

3.  Hydrochloric  acid  of  0.6  per  cent,  may  easily  be  prepared  from  cone, 
hydrochloric  acid  (36.5  per  cent.)  by  diluting  two  cc.  of  the  cone,  acid  to 
120  cc.  with  water. 


GASTRIC  ANALYSIS  9 

In  carcinoma  values  as  low  as  three  may  be  observed  or  in  fact 
there  may  be  no  digestion  at  all. 

12.  Rennin. — Three  to  five  drops  of  the  gastric  contents  are 
added  to  10  cc.  of  milk,  and  the  mixture  warmed  to  about  35°  C, 
either  in  a  water-bath  or  in  an  incubator.  If  coagulation  takes 
place  in  fifteen  minutes  rennin  is  present  in  moderate  amounts. 

13.  Products  of  Digestion. — Proteoses  and  peptones  may  be 
inferred  in  the  presence  of  free  HC1  and  pepsin,  and  the  extent  of 
starch  digestion  may  be  ascertained  with  dilute  iodine  and  Bene- 
dict's solution. 

14.  Blood. — If  the  microscope  has  not  revealed  erythrocytes, 
the  guaiac  or  other  chemical  tests  may  be  applied  preferably 
upon  the  retention  when  a  retention  meal  has  also  been  employed. 
To  a  small  amount  of  the  sediment,  add  an  equal  volume  of  30 
per  cent,  acetic  acid  and  extract  with  ether.  If  the  blood  is 
present  in  considerable  amount,  the  ether  will  assume  a  brown- 
ish-red color.  Filter  off  the  ether  extract  and  to  a  portion  of  the 
filtrate,  add  10  drops  of  an  alcoholic  solution  of  guaiac1  and  20 
to  30  drops  of  old  turpentine  or  hydrogen  peroxide.  In  the  pres- 
ence of  blood,  a  blue  color  is  produced.  Other  reagents,  as 
benzidine,  phenol phthalin  or  aloin  may  be  employed,2  but  this 
test  is  very  satisfactory  and  sufficiently  delicate. 

15.  Diagnostic  Tests  for  Carcinoma  of  the  Stomach. — Various 
tests  have  been  proposed  as  specific  for  carcinoma  of  the  stomach, 
but  none  of  these  tests  can  be  said  to  have  been  found  entirely 
trustworthy.  Futhermore,  these  tests  do  not  react  positively 
until  the  disease  is  quite  well  developed  clinically.  Two  tests 
which  are  perhaps  worthy  of  mention  are  the  Salomon  test  and 
the  Neubauer-Fischer  tryptophane  test. 

a.  Salomon  Test*. — The  principle  underlying  this  test  is 
the  fact  that  carcinomata  secrete  protein,  which  becomes 
mixed  with  the  gastric  contents.  The  diet  of  the  patient 
for  24  hours  prior  to  the  test  should  be  free  from  soluble 
protein.  At  the  beginning  of  this  period  he  is  given  a  morn- 
ing meal  of  milk  and  gruel  and  a  mid-day  meal  of  boullion 
with  coffee  or  tea.  Late  in  the  evening,  the  stomach  should 
be    washed    out    with    large    quantities    of    pure   water    until 

1.  One  gram  of  good  guaiac  resin  dissolved  in  60  cc.  of  95  per  cent, 
alcohol. 

2.  See  Chapter  II,   p.   24. 

3.  Salomon:   Deutsch.  med.   Wochschr.,  1903,  XXIX,  p.  547. 


10  PATHOLOGICAL  CHEMISTRY 

the  return  water  is  clear.  The  following  morning  the  fast- 
ing stomach  is  washed  twice  with  400  cc.  of  physiological  salt 
solution,  the  same  solution  being  used  each  time.  The  total 
nitrogen  and  protein  are  estimated  in  this  wash  water  by 
the  Kjeldahl  and  Esbach  methods  respectively.  (For 
methods,  see  chapters  on  urine).  Salomon  found  in  cases 
of  gastric  carcinoma,  *  20  to  70  mgms.  of  nitrogen  and  from 
0.00625  to  0.05  gms.  protein  to  each  100  cc.  of  fluid.  In 
non -malignant  cases,  no  protein  was  found,  and  the  nitrogen 
varied  from  0  to  16  mgms. 

b.  Tryptophane  Test— LThis  test  was  first  suggested  by 
Neubauer  and  Fischer1  with  the  use  of  glycyltryptophane. 
Weinstein2  entirely  dispensed  with  the  expensive  glycyltryp- 
tophane, and  simply  tested  the  juice  obtained  after  a  regular 
meal.  This  technique  has  been  found  to  be  less  reliable  than  the 
original  test,  though  Jacque  and  Woody att3  have  recently  ob- 
served that  a  solution  of  Witte's  peptone  is  nearly  as  efficient  as 
glycyltryptophane.  The  modified  procedure  is  as  follows: 
Four  to  five  hours  after  a  regular  meal  some  stomach  contents  are 
secured  and  filtered.  About  5  cc.  of  the  juice  are  then  mixed  with 
an  equal  volume  of  sterile  2  per  cent.  Witte's  peptone,  toluene 
added,  the  tube  shaken,  and  incubated  at  body  temperature 
for  24  hours.  At  the  end  of  this  time  three  to  four  cc.  of  the 
mixture  are  taken  and  if  not  acid,  treated  with  a  few  drops  of 
three  per  cent,  acetic  acid.  Bromine  water4  is  now  added  drop 
by  drop,  until  in  case  the  reaction  is  positive,  a  reddish  violet 
,color  appears. 

This  te§t  is  based  on  the  fact  that  carcinomatous  tissue  con- 
tains an  enzyme  of  stronger  proteolytic  power  than  pepsin,  caus- 
ing the  appearance  of  amino  acids,  including  tryptophane. 
Blood  and  trypsin  must  not  be  present.  (The  latter  may  be 
regarded  as  absent  if  bile  has  not  been  detected) . 

Smithies5  in  the  Mayo  Clinic  has  found  that  more  than  one- 
third  of  the  proved  cases  of  cancer  of  the  stomach  gave  the 
glycyltryptophane  test,  more  than  one  fourth  were  lactic  acid 

1.  Neubauer  and  Fischer:     Deutsch.  Atch.  f.  klin.  Med.,  1909,  XCVII, 

p.  499. 

2.  Weinstein:     Jour.  Amer.  Med.  Assoc,  1910,  LV,  p.  1085. 

3.  Jacque  and  Woodyatt:      Arch.  Int.  Med.,  1912,  X,  p.  560. 

4.  Water  to  which  sufficient  bromine  has    been  added  to  saturate  it. 

5.  Smithies:     Arch.  Int.  Med.,  1912,  X,  p.  357. 


GASTRIC  ANALYSIS  11 

positive,  while  about  one-thirteenth  gave  the  Weinstein  trypto- 
phane test. 

16.  Microscopical  Examination. — This  is  to  be  made  upon 
the  sediment  of  the  retention  meal  or  better  still  upon  fluid  re- 
moved from  the  fasting  stomach,  looking  particularly  for  leu- 
cocytes, erythrocytes,  starch  grains,  bacilli  (especially  the  Boas- 
Oppler  bacillus)  and  tumor  particles. 


CHAPTER  II. 

DIGESTIVE   CHANGES    IN   THE   INTESTINE,    TOGETHER  WITH 

THE  FORMATION,    COMPOSITION    AND    CLINICAL 

SIGNIFICANCE  OF  THE  FECES. 

It  will  be  recalled  that  food  materials  which  have  passed 
the  pyloric  sphincter  into  the  duodenum,  are  normally  composed 
of  the  proteins,  quite  largely  in  the  proteose -peptone  stage  of 
hydrolysis,  the  starchy  material,  in  the  dextrin  stage,  while 
the  fatty  foods  have  encountered  practically  no  change.  The 
hydrochloric  acid  of  the  chyme,  when  present  in.  the  duodenum, 
acts  upon  a  substance  termed  prosecretin,  transforming  it  into 
secretin,  and  the  latter,  through  the  medium  of  the  blood, 
stimulates  a  discharge  of  bile  and  a  secretion  of  pancreatic 
juice.  The  pancreatic  juice  is  an  alkaline  fluid  and  contains 
amylolytic,  lipolytic,  and  proteolytic  enzymes,  i.e.,  enzymes  which 
act  upon  all  three  types  of  food  stuffs.  The  amylopsin  of  the 
pancreas  has  a  stronger  action  upon  starch  digestion  than  the 
ptyalin,  and  converts  any  unattacked  starch  and  the  dextrins 
to  maltose.  Likewise  the  trypsinogen,  after  its  activation  by 
the  enterokinase  of  the  intestinal  juice,  breaks  down  any  un- 
altered protein  into  proteoses  and  peptones,  and  to  a  considerable 
extent,  splits  these  up  into  their  constituent  amino  acids.  The 
action  of  the  pancreatic  lipase  is  likewise  one  of  hydrolytic  cleav- 
age, in  which  the  fats  are  split  into  their  simplest  components, 
the  fatty  acids  and  glycerol,  in  which  transformation  the  lipase 
is  greatly  facilitated  by  the  bile  salts.  Digestion  has  now  by 
no  means  been  completed.  In  the  secretion  of  the  intestinal 
juices,  enzymes  are  to  be  found  which  carry  out  many  of  the 
final  transformations.  Before  they  are  ready  for  absorption,  the 
disaccharides,  maltose,  lactose  and  sucrose,  must  be  broken 
down  to  the  monosaccharides.  In  the  succus  entericus  are  found 
the  enzymes,  maltase,  lactase  and  su erase,  for  this  purpose.  The 
proteoses  and  peptones  which  have  escaped  complete  hydrolysis 
by  the  trypsin  will  find  the  enzyme,  erepsin,  waiting  to  complete 
this  action.     Thus  we  observe  that  digestion  really  consists  in 

12 


COMPOSITION  OF  FECES  13 

the  breaking  down  of  our  various  food  materials  into  the  smallest 
possible  integral  parts  or  "  Bausteine."  From  these,  our  body- 
carbohydrate,  fat  and  protein  are  constructed.  The  monosac- 
charides, of  which  glucose  is  the  chief,  are  picked  up  by  the  portal 
circulation  and  carried  to  the  liver,  where  the  sugar,  over  and 
above  approximately  one  tenth  of  one  per  cent,  is  stored  up  as 
glycogen.  The  recent  researches  of  Folin  make  it  evident  that 
the  amino  acids  themselves  are  picked  up  by  the  blood,  but  just 
where  their  synthesis  into  body  protein,  or  the  deamidization  of 
the  acids  not  employed  in  tissue  repair  takes  place  is  not  clear. 
Fat  appears  to  be  reformed  from  the  fatty  acids  and  glycerol  by 
the  epithelial  cells  of  the  intestinal  wall  which  absorb  these  cleav- 
age products.  It  is  then  picked  up  by  the  lacteals,  and,  in  the 
form  of  a  very  fine  emulsion  (chyle) ,  carried  by  the  lymphatics 
to  the  thoracic  duct,  and  there  emptied  into  the  blood  stream. 

The  food  materials  are  propelled  along  the  intestine  by  the 
same  peristaltic  movements  that  have  previously  been  noted  in 
the  esophagus  and  stomach.  A  series  of  advancing  waves  of 
constriction,  preceded  by  waves  of  relaxation,  serve  to  propel 
the  masses  of  food  material.  In  addition  to  the  peristaltic  wave, 
a  second  type  of  movement  is  observed  in  the  small  intestines, 
viz.,  a  series  of  local  constrictions  occurring  rhythmically  at 
those  points  at  which  masses  of  food  lie.  The  apparent  purpose 
of  this  is  to  thoroughly  mix  the  material  with  the  digestive 
secretions  and  bring  it  in  intimate  contact  with  the  absorptive 
walls.  The  consistency  of  the  fluid  passing  the  ileocecal  valve  is 
about  the  same  as  that  leaving  the  stomach.  The  greater  part 
of  the  nutritive  materials  are  absorbed  while  they  are  being  passed 
along  the  small  intestines.  The  absorption  of  the  excess  of 
water  takes  place  in  the  colon,  however,  and  is  aided  by  waves  of 
antiperistalsis  passing  at  intervals  over  the  ascending  portion. 
In  the  descending  colon  peristaltic  waves  are  again  observed 
which  carry  the  feces  toward  the  rectum. 

The  reaction  of  food  material  as  it  passes  through  the  body 
undergoes  many  changes.  The  strongly  acid  character  of  the 
chyme,  as  it  leaves  the  pyloric  sphincter,  is  soon  changed  to  a 
faint  alkalinity  by  the  alkaline  pancreatic  juice.  However, 
when  the  reaction  of  the  fluid  passing  the  ileocecal  valve  is  taken, 
it  will  be  found  to  be  slightly  acid,  due  to  bacterial  fermentation, 
but  again  bacterial  changes  in  the  colon  render  the  material 


14  PATHOLOGICAL  CHEMISTRY 

discharged  from  *  the  rectum  neutral  or  even  faintly  alkaline  to 
litmus. 

The  fact  that  about  one- third  of  the  dry  matter  of  normal 
human  feces  consists  of  bacteria,  and  at  least  one-half  of  the  nitro- 
gen of  the  feces  is  bacterial  in  its  origin1  serves  to  emphasize 
the  importance  of  bacteria  in  the  intestinal  canal,  though  experi- 
mental evidence  would  indicate  that  the  presence  of  this  large 
number  of  bacteria  is  a  normal  and  even  useful  condition. 
In  nurslings,  the  bacterial  flora  is  relatively  simple,  though 
later  in  life,  the  number  of  these  bacterial  forms  becomes  very 
large.  The  dominant  organism  in  nurslings  is  B.  bifidus,  but 
this  is  ultimately  replaced  by  B.  coli  and  B.  lactis  aero  genes. 
Other  organisms  which  may  be  observed  are  coccal  forms,  B. 
aerogenes  capsulatus,  and,  in  certain  cases,  B.  putrificus.  These 
lasc  two  organisms  Herter2  is  inclined  to  associate  with  con- 
dicions  of  excessive  putrefaction  in  the  intestines.  In  early  life, 
the  products  of  intestinal  decomposition  are  remarkably  small 
in  amount,  and,  as  would  be  expected,  the  number  of  putre- 
factive bacteria  are  few.  One  finds,  however,  in  middle  life  a  large 
number  of  persons  in  whom  the  putrefactive  conditions  in  the 
intestine  are  distinctly  more  active  than  was  the  case  earlier  in 
life.  Unquestionably  the  most  important  factors  in  bringing 
about  this  strongly  proteolyzing  type  of  bacterial  flora  are  the 
consumption  of  an  overabundance  of  protein  food,  combined  with 
inadequacy  in  the  digestive  juices,  delayed  absorption,  and  in- 
sufficient motility  in  the  alimentary  canal.  Very  little  decom- 
position takes  place  in  the  large  intestine  under  the  action  of 
B.  coli,  if  the  absorption  in  the  small  intestine  has  been  good. 

It  is  easy  to  comprehend  that  urinary  constituents,  such  as  urea, 
uric  acid,  creatinine,  etc.  are  derived  from  the  metabolism  of  pro- 
tein in  the  body,  whether  the  protein  be  the  body's  own,  or  that  of 
an  animal  fed  to  it,  but  the  intestinal  canal,  where  the  feces  are 
formed,  is  a  long  tube  open  at  both  ends,  through  which  may  pass 
the  nitrogen  gas  of  the  air  swallowed  and  various  indigestible  ma- 
terials. In  diarrhea,  the  curds  of  milk,  pieces  of  undigested  meat 
or  bread,  and  large  quantities  of  fat  are  in  evidence.     These  com- 

1.  Schmidt  and  Strasburger:  Die  Fazes  des  Menschen,  3rd.  edit., 
Berlin,  1910,  p.  327;  MacNeal,  Latzer  and  Kerr:  Jour,  Inf.  Dis.,  1909,  VI., 
p.  123;  Matill  and  Hawk:  Arch.  Int.  Med.,  1911,  XIV.,  p.  433. 

2.  Herter:  The  Common  Bacterial  Infections  of  the  Digestive  Tract, 
1907;  Cf.  also  MacNeal,  Latzer  and  Kerr:  Jour.  Inf.  Dis.,  1909, VI.,  p.  571. 


COMPOSITION  OF  FECES  15 

mon  observations  would  seem  to  justify  the  general  supposition 
that  normal  feces  are  made  up  of  undigested  food  residues.  On  the 
contrary,  this  is  far  from  the  fact.  The  feces  are  chiefly  the 
unabsorbed  residues  of  intestinal  secretions.  Furthermore,  as 
Mendel  and  others  have  shown,  the  feces  is  the  normal  path 
for  the  elimination  of  a  number  of  the  important  inorganic  ele- 
ments, such  as  iron,  calcium,  etc.  As  a  proof  that  feces  are  a 
true  secretion,  it  has  been  shown  by  F.  Voit  that  the  material 
secreted  in  an  isolated  loop  of  the  intestine  of  a  dog  is  of  a  similar 
composition,  and  contains  the  same  amount  of  nitrogen  as  the 
feces  of  the  normal  intestine  through  which  food  is  passing. 
Prausnitz  defines  normal  feces  as  those  resulting  from  the  eating 
of  any  food  that  is  completely  digested  and  absorbed.  It  is 
entirely  probable  that  on  a  diet  whose  constituents  are  not 
entirely  available,  the  amount  of  feces  is  increased  by  the  undi- 
gested cellulose,  and  the  nitrogen  content  is  increased  by  the 
large  amount  of  digestive  juices  secreted,  because  of  the  large 
volume  of  food  and  the  accompanying  increased  peristalsis.  As 
pointed  out  above,  a  large  part  of  the  dry  matter  of  human  feces 
consists  of  bacterial  substance.  A  larger  part  of  the  organic 
material  eliminated  in  the  feces  is,  however,  of  unknown  nature 
and  composition. 

Amount. — Upon  an  ordinary  mixed  diet,  the  daily  fecal  ex- 
cretion of  an  adult  male  will  average  110  to  170  grams,  with  a 
solid  content  varying  between  25  and  45  grams.  The  feces  of 
such  an  individual  upon  a  vegetable  diet  will  be  much  greater 
and  may  even  amount  to  -350  grams  and  possess  a  solid 
content  of  75  grams.  The  variation  in  the  normal  daily  output 
being  so  great  renders  this  factor  of  little  value  for  diagnostic 
purposes,  except  where  the  composition  of  the  diet  is  accurately 
known.  Lesions  of  the  digestive  tract,  a  defective  absorptive 
function,  or  increased  peristalsis,  as  well  as  admixture  of  mucus, 
pus,  blood  and  pathological  products  of  the  intestinal  wall  may 
cause  the  total  amount  of  excrement  to  be  markedly  increased. 

Consistency. — The  form  and  consistency  of  the  stool  is  de- 
pendent, in  large  measure,  upon  the  nature  of  the  diet.  Under 
normal  conditions  the  consistency  may  vary  from  a  thin,  pasty 
discharge  to  a  firmly  formed  stool.  Stools  which  are  exceedingly 
thin  and  watery  ordinarily  have  a  pathological  significance. 

Color. — The  fecal  pigment  of  the  normal  adult  is  hydrbbili- 
rubin,  also  called  stercobilin.     It  originates  from  the  bilirubin 


16  PATHOLOGICAL  CHEMISTRY 

which  is  secreted  into  the  intestine  in  the  bile,  being  formed  by 
the  reducing  activity  of  certain  bacteria.  Hydrobilirubin  bears 
a  close  resemblance  to  urobilin  and  may  even  be  identical  with 
that  pigment.  Neither  bilirubin  nor  biliverdin  occurs  normally 
in  the  fecal  discharge  of  adults,  although  the  former  may  be  de- 
tected in  the  excrement  of  nursing  infants.  The  diet  is  the  most 
important  factor  in  determining  the  color  of  the  fecal  discharge. 
A  mixed  diet,  for  instance,  produces  stools  which  vary  in  color 
from  light  to  dark  brown;  an  exclusive  meat  diet  gives  rise  to  a 
brownish  black  stool,  whereas  the  stool  resulting  from  a  milk 
diet  is  invariably  light  colored.  That  certain  drugs  act  to  color 
the  fecal  discharge  is  well  illustrated  by  the  occurrence  of  green 
stools  following  the  use  of  calomel,  and  of  black  stools  after 
bismuth  ingestion. 

Odor. — The  odor  of  normal  feces  is  generally  stated  to  be  due 
to  skatole  and  indole.  However,  these  aromatic  putrefactive 
substances  are  generally  found  in  such  small  amounts  as  to  form 
an  insufficient  explanation  on  this  point.  Hydrogen  sulphide 
no  doubt  plays  a  certain  part  in  the  disagreeable  character  of  the 
odor.  The  intensity  of  the  odor  depends  to  a  large  degree  upon 
the  kind  of  diet,  being  very  marked  in  stools  from  a  meal 
diet,  much  less  marked  in  stools  from  a  vegetable  diet,  and  fre- 
quently hardly  detectable  in  stools  from  a  milk  diet.  Thus  the 
stool  of  the  infant  is  ordinarily  odorless,  and  any  decided  odor 
may  generally  be  traced  to  some  pathological  source. 

Reaction. — Normal  stools  generally  have  a  neutral  reaction, 
although  slightly  alkaline  or  even  acid  stools  are  met  with.  The 
acid  reaction  is  encountered  much  less  frequently  than  the  alka- 
line and  then  commonly  only  following  a  vegetable  diet. 

A  simple  division  of  fecal  material  may  be  based  upon  the 
separation  afforded  by  the  customary  procedures,  viz.,  the  esti- 
mation of  the  total  nitrogen,  ethereal  extract,  and  carbohydrate 
residues.  The  results  obtained  with  these  methods  have  yielded 
data  of  great  scientific  importance,  though  the  time  required 
and  the  nature  of  the  results  obtained  often  render  them  of 
rather  small  value  diagnostically. 

Nitrogenous  Substances. — Three  sources  are  usually  considered 
as  contributing  to  the  nitrogenous  material  excreted  in  the  feces ; 
food  residues,  residues  of  the  digestive  juices  and  cellular  material 
from  the  intestinal  wall,  and  bacteria  and  their  products.     The 


COMPOSITION  OF  FECES  17 

quantity  of  this  nitrogen  amounts  to  from  one:half  to  two  grams 
and  from  four  to  eight  per  cent,  of  the  dry  feces.  Upon  a  meat 
diet  the  food  residues  represent  almost  nothing  under  normal  con- 
ditions, i.e.,  the  muscle  protein  is  practically  100  per  cent,  util- 
ized1. In  the  case  of  vegetable  proteins  it  has  been  a  matter 
of  common  observation  that  the  utilization  was  not  as  good 
as  with  animal  proteins.  This  in  part  at  least  is  explained 
by  the  inaccessability  of  certain  of  the  vegetable  proteins  to  the 
digestive  juices,  for  as  Mendel  and  Fine2  have  shown,  the  proteins 
of  the  wheat,  and  probably  also  of  the  barley  and  corn  are  as  well 
utilized  as  meat,  when  taken  in  a  pure  form  or  freed  from  extrane- 
ous cellular  substance.  With  legumes  the  utilization  was  not  quite 
as  good,  though  this  point  they  are  as  yet  unable  to  fully  explain. 
That  about  one-half  the  fecal  nitrogen  is  derived  from  the  fecal 
bacteria  has  already  been  pointed  out.  A  great  variety  of 
nitrogenous  substances  may  be  formed  by  bacterial  action  upon 
the  protein  or  its  cleavage  products.  Among  such  may  be 
mentioned  indole,  skatole,  phenol,  indole  acetic  acid,  various 
oxy-acids,  in  certain  instances  putrescine  and  cadaverine,  etc. 
That  intoxication  may  result  from  poisonous  products  formed 
by  bacterial  action  can  hardly  be  questioned,  though  just  what 
the  substances  are  that  exert  this  effect  cannot  be  stated  at  the 
present  time.3  Much  attention  has  been  devoted  to  the  pro- 
ducts of  bacterial  action  on  tryptophane,-  viz.,  indole  acetic 
acid  (urorosein),  skatole  and  indole.  The  presence  of  a  large 
amount  of  indican  in  the  urine  is  no  doubt  indicative  of  increased 
intestinal  putrefaction.  It  is  questionable,  however,  whether 
indole  in  the  amounts  absorbed  in  this  way  has  any  toxic 
properties. 

With  regard  to  the  elimination  of  fecal  nitrogen  under  patho- 
logical conditions,  observations4  show  that  it  is  increased  in 
biliary  obstruction,  intestinal  fermentative  dyspepsia,  and 
diarrhea;  and  decreased  in  chronic  constipation. 

Ethereal  Extract. — The  bodies  which  go  to  make  up  this  ethe- 
real extract  are  the  neutral  fats,  free  fatty  acids,  (and  fatty  acids 

1.  Cf.  Mendel  and  Fine:     Jour.  Biol.  Chem.,  1912,  XI,  p.  22. 

2.  Mendel  and  Fine:  Jour.  Biol.  Chem.,  1911,  X,  pp.  303,  399,345, 
433. 

3.  Myers,  Fisher  and  Diefendorf :  Amer.  Jour.  Insanity,  1909,  LXV,  p. 
607;  also  Zentr.  f.  Stoffwechsels,  1908,  IX.,  p.  849. 

4.  Schmidt  und  Strasburger;   Die  Fazes  des  Menschen,  p.  130. 


18  PATHOLOGICAL  CHEMISTRY 

in  the  form  of  soaps  when  an  acidified  solvent  has  been  employed) , 
and  koprosterol  formed  from  cholesterol  by  the  action  of  reduc- 
ing bacteria.  This  ethereal  extract  ordinarily  forms  from  12 
to  25  per  cent,  of  the  dry  weight  of  the  feces.  The  util- 
ization of  fat  varies  under  normal  conditions  from  90  to  95 
per  cent,  depending  upon  the  source  of  food.  The  higher 
fats  such  as  stearin  are  much  less  readily  assimilated.  In 
biliary  obstruction  as  much  as  70  grams  of  fat  may  be  eliminated 
in  the  feces,  forming  50  per  cent,  of  the  dry  weight  of  the  material. 
In  various  conditions  associated  with  defective  fat  digestion 
(pancreatic  disease)  or  defective  fat  absorption  increased 
amounts  may  be  eliminated,  while  in  chronic  constipation  the 
amount  may  be  decreased.  In  both  biliary  obstruction  and 
pancreatic  disease  the  fat  utilization  has  been  found  to  be  as 
low  as  25  per  cent. 

Carbohydrate  Residues. — Normally  feces  may  yield  on  hy- 
drolysis reducing  substance  equivalent  to  from  one-half  to  two 
grams  of  glucose  or  from  two  to  six  per  cent,  of  the  dry  weight  of 
the  feces.  The  utilization  of  the  carbohydrate  is  regarded  to  be 
about  98  per  cent.  Ordinarily  starch  digestion  does  not  seem  to 
be  interfered  with,  though  the  amount  of  carbohydrate  material 
eliminated  in  the  severer  catarrhal  conditions  of  the  intestine 
may  be  slightly  increased.  One  question  to  be  asked  with  regard 
to  all  carbohydrate  material  is,  are  the  enzymes  of  the  alimentary 
canal  capable  of  hydrolyzing  it.  There  appear  to  be  no  enzymes 
in  the  digestive  tract  capable  of  attacking  certain  of  the  more 
complex  carbohydrates1. 

To  determine  the  functional  capacity  of  the  intestine,  a  test- 
diet  may  be  administered  on  the  same  general  principles  as  this 
is  employed  in  connection  with  gastric  activity.  The  following 
test-diet  has  been  suggested  by  Schmidt  and  Strasburger2. 

In  the  morning,  one-half  liter  of  milk  (or  tea,  or  cocoa,  if  pos- 
sible with  much  milk)  together  with  one  roll  and  butter,  and  one 
soft-boiled  egg;  for  breakfast,  one  dish  of  oatmeal-gruel,  cooked 
in  milk  and  strained  (salt  or  sugar  permissible);  at  noon,  one- 
quarter  pound  finely  chopped  lean  beef,  broiled  rare,  with  butter, 


1.  Mendel:    Zentr.  f.  Stoffwechsels,  1908,  IX,  p.  641. 

2.  Schmidt  and  Strasburger:  Die  Fazes  des  Menschen,  1910,  also 
Schmidt:  The  Examinations  of  the  Function  of  the  Intestines  by  Means 
of  the  Test  Diet,  2nd  Amer.  Edit,  by  Aaron,  Phila.,  1909,  p.  11. 


COMPOSITION  OF  FECES  19 

the  interior  raw,  along  with  it,  not  too  small  a  portion  of  potato 
broth  (well  strained) ;  in  the  afternoon,  the  same  as  in  the  morning 
but  no  egg;  in  the  evening,  one-half  liter  of  milk,  or  one  plate  of 
soup  (as  in  the  morning),  together  with  a  roll  and  butter,  and 
one  or  two  soft  boiled  eggs. 

The  diet  in  a  healthy  individual  should  yield  the  normal  feces, 
according  to  the  idea  of  Prausnitz,  i.e.,  it  should  have  practically 
no  food  residues  and  be  almost  entirely  "  metabolic  "  in  origin. 
This  diet  may  be  varied  slightly,  depending  upon  the  information 
desired.  The  essential  ingredients  are  the  milk,  white  bread, 
potato  broth  and  half-raw  meat  to  show  various  possible  de- 
ficiencies. The  diet  employed  in  this  hospital  consists  of  the 
following:  for  breakfast,  tea  or  coffee  with  cream,  farina  and  two 
eggs;  for  lunch,  chopped  meat  cooked  as  above,  mashed  potatoes 
and  white  bread;  for  dinner,  rice  or  macaroni,  toast  or  zweibach 
with  butter,  milk  and  one  or  two  eggs. 

For  quantitative  work,  a  detailed  test-diet,  such  as  that 
originally  suggested  by  Schmidt  and  Strasburger1  which  con- 
tains about  110  grams  protein,  105  grams  fat  and  200  grams 
carbohydrate,  should  be  employed.  For  making  the  functional 
test,  the  patient  is  kept  on  the  diet  for  three  days,  or  even  longer, 
until  a  stool  is  obtained  which  is  certainly  derived  from  it. 
Under  normal  conditions,  this  occurs  at  the  second  defecation 
after  the  beginning  of  the  test.  To  mark  this  point,  the  patient 
must  take  a  five  grain  capsule  of  willow  charcoal.  This  substance 
passes  through  the  intestine  unaltered  and  marks  off  the  cor- 
responding point  in  the  feces.  In  this  way,  also  both  the  begin- 
ning and  the  end  of  a  period  may  be  determined,  so  that  if  feces 
are  desired  for  the  whole  of  a  period,  for  example,  three  days, 
they  may  easily  be  separated. 

Many  of  the  valuable  hints  obtained  from  the  examination  of 
the  feces  following  the  test  diet  are  mentioned  in  connection  with 
the  laboratory  tests.  Some  of  the  more  salient  features  will 
bear  discussion  here.  The  appearance  of  mucus  in  the  feces 
indicates  the  existence  of  an  inflammatory  condition  of  the  mu- 
cous membrane.  Fatty  stools  may  have  their  origin  in  a  biliary 
obstruction  or  deficiency,  a  disturbance  of  pancreatic  secretion, 
or  a  disturbance  of  intestinal   digestion.2      If  connective   tissue- 

1.  Cf.  Schmidt- Aaron:  Loc.  cit.,  p.  13. 

2.  Schmidt-Aaron:  Loc.  cit.,  p.  39. 


20  PATHOLOGICAL  CHEMISTRY 

remains  appear  in  the  feces,  it  is  a  sign  of  disturbance  in  gastric 
digestion.  The  gastric  juice  alone  can  digest  raw  connective 
tissue.  If  macroscopic  muscle-remains  appear  in  the  feces,  this 
is  a  sign  of  a  disturbance  of  digestion  in  the  small  intestines. 
Many  valuable  hints  with  regard  to  carbohydrate  and  also 
protein  remains  are  obtained  from  the  incubator  test. 

The  so-called  nuclei  test  of  Schmidt  is  perhaps  as  reliable  a 
test  for  pancreatic  inefficiency  as  any  we  possess.  One-half  cm. 
cubes  of  beef  or  thymus1  are  hardened  in  alcohol,  but  previous 
to  use  washed  for  several  hours  in  water,  then  tied  in  a  silk 
gauze  bag,  and  served  with  the  test  diet.  If  pancreatic  secre- 
tion is  suspended,  nuclei  will  be  found  in  the  silk  gauze  bag 
which  appears  in  the  stool,  and  can  easily  be  recognized  with  the 
aid  of  the  microscope. 

Laboratory  Procedures 

Feces  may  be  very  conveniently  collected  for  examination  in 
glass,  screw  top  jars  about  four  inches  high  and  four  inches  in  dia- 
meter. If  the  purpose  of  the  examination  is  for  the  functional 
examination  of  the  alimentary  tract,  the  feces  should  be  obtained 
after  a  diet  such  as  that  of  Schmidt  and  Strasburger  mentioned 
above.  All  specimens  should  be  examined  as  soon  as  possible 
after  evacuation. 

1.  Macroscopic  Examination. — This  forms  the  most  import- 
ant part  of  the  whole  procedure,  and  alone  is  often  sufficient  to 
enable  the  experienced  observer  to  form  a  judgment.  In  the 
first  place,  it  determines  whether  color,  consistency  and  odor 
correspond  with  the  normal  feces. 

a.  Color. — Milk  produces  light  brown  feces;  cocoa,  red- 
dish brown;  absence  of  bile,  or  any  condition  producing  a 
large  amount  of  fat,  gives  clay-colored  stools;  blood  from  the 
upper  part  of  the  alimentary  tract  yields  "  tar  feces."  Fer- 
mentation feces  are  light  brown  and  foamy,  while  putre- 
factive feces  are  dark. 

b.  Odor. — Mildly  excremental  (normal) ;  butyric  acid-like 
(fermentation) ;  malodorous  (putrefactive) . 

1.  The  use  of  thymus  cubes  was  suggested  by  Einhorn  and  because  of 
the  great  abundance  of  nuclei  is  to  be  preferred  to  the  beef.  Further  with 
Einhorn's  bead  test,  the  test  diet  may  be  entirely  dispensed  with.  Einhorn 
discusses  the  important  facts  connected  with  his  test  in  a  recent  number 
of  the  Post-Graduate,  May  1912,  XXVII.,  p.  359. 


ANALYSIS  OF  FECES  21 

c.  Consistency. — The  feces  may  be  hard,  well-formed, 
with  wide  bore;  thin  and  soft;  soft  with  admixture  of  hard 
lumps;  diarrheal;  they  may  contain  mucus,  intimately 
mixed  with  feces,  or  easily  separated  from  the  same;  un- 
digested matter,  e.g.,  fruit  stones,  vegetable  skins,  intestinal 
gravel,  gall-stones,  parasites,  etc. 

Under  pathological  conditions,  such  food-remains  as  the  follow- 
ing may  appear:  remains  of  connective  tissue  and  tendons  from 
the  chopped  meat  eaten,  remains  of  muscle  tissue,  potato  remains 
(glassy,  transparent  granules,  which  appear  like  sago-grain), 
and  fat  remains.  Other  consitutents  which  may  be  observed 
are:  mucus,  in  large  and  small  flakes,  and  large  crystals  of  am- 
monium-magnesium phosphate. 

2.  Micro  chemical  Examination. — This  serves  for  the  most  part 
to  supplement  the  macroscopic  examination.  The  feces  are  thor- 
oughly mixed,  and  a  portion  as  large  as  a  walnut  transferred  to 
a  mortar  and  ground  with  water  to  a  thin  mush.  A  drop  is 
transferred  to  each  of  four  slides,  or  the  four  drops  may  be 
placed  upon  the  four  parts  of  the  same  slide.  Both  the  low 
(!)  and  high  (f)  power  objectives  are  employed  in  this 
examination. 

a.  The  first  drop  is  simply  covered  with  a  cover  glass.  It 
is  examined  for  muscle  fibers,  noting  their  relative  abund- 
ance and  state  of  preservation,  connective  tissue  remains, 
yellow  fatty  acid  salts  of  calcium,  colorless  soaps,  drops  of 
neutral  fat,  mucus  particles,  leucocytes,  erythrocytes,  vege- 
table cells,  etc. 

b.  The  second  drop  is  throughly  mixed  with  a  drop  of 
36  per  cent,  acetic  acid  and  heated  until  bubbles  appear.  It 
is  covered  with  a  cover  glass  and  examined  for  fatty  acid 
flakes.  This  treatment  also  serves  to  differentiate  between 
connective  tissue  remnants  and  mucus,  the  former  being 
rendered  transparent. 

C.  The  third  drop  is  treated  with  a  couple  of  drops  of 
a  saturated  70  per  cent,  alcoholic  solution  of  Sudan  III, 
which  stains  the  fat  globules  red. 

d.  The  fourth  drop  is  rubbed  up  with  a  drop  of  iodine 
solution.1  It  is  examined  for  starch  granules,  fungi,  yeast 
cells,  etc.  (colored  blue  or  violet). 

1.  The  iodine  solution  is  prepared  by  adding  sufficient  iodine  to  a  2 
per  cent,  potassium  iodide  solution  to  color  it  a  light  brown. 


22  PATHOLOGICAL  CHEMISTRY 

The  following  conditions  are  pathologic:  In  "  a,"  fragments 
of  muscle  in  large  numbers  and  good  state  of  preservation,  needles 
of  fatty  acid  and  soap,  drops  of  neutral  fat,  numerous  groups 
of  potato  cells,  parasite  eggs,  mucus,  connective  tissue,  pus,  etc.; 
in  "  b,"  massive  fatty  acid  flakes;  in  "  c,"  many  red  fat  globules, 
and  in  "  d,"  many  blue  colored  remains  of  starch  granules,  blue 
or  violet  fungous  spores  and  yellow  yeast  cells. 

3.  Reaction. — The  reaction  is  taken  with  strips  of  red  and 
blue  litmus  paper  upon  feces  ground  up  with  distilled  water. 

4.  Schmidt's  Sublimate  Test. — Some  feces  rubbed  up  with 
water,  are  placed  in  saturated  mercuric  chloride,  stirred,  and 
allowed  to  stand  over  night.  Normal  feces  are  colored  red 
(hydrobilirubin.)  A  green  color  (even  if  confined  to  microscop- 
ical particles)  is  pathological  (unchanged  bilirubin).  In  oc- 
clusion of  the  bile  duct,  no  color  is  obtained. 

5.  Fermentation  Test. — From  the  fresh  excrement,  which  has 
been  stirred  up,  but  not  yet  thinned  with  water,  a  small  amount, 
about  the  size  of  a  walnut,  is  taken  with  a  wooden  spatula, 
and  put  into  the  lower  vessel  a  of  Strasburger's  fermentation 
tube.1  (In  hard  stools  less  is  taken;  in  soft,  more;  in  fluid  stools, 
the  lower  vessel  is  entirely  filled).  In  this,  it  is  stirred  up  with 
water  with  the  aid  of  a  wooden  spatula,  and  then  the  rubber 
stopper  is  put  on,  care  being  taken  to  exclude  air  bubbles.  The 
rubber  stopper  is  now  taken  from  the  little  tube  b  and  the  tube 
filled  with  tap-water.  This  is  then  closed  by  turning  over 
the  vessels  a  together  with  the  small  stoppers,  and  the 
vessel  c  (empty)  connected  with  it;  c  has  an  opening  on  top  and 
acts  as  an  ascension  pipe.  When  the  apparatus  is  put  together, 
it  is  placed  for  24  hours  in  an  incubator  heated  to  37°  C. 
If  gas  is  developed  from  the  feces  within  this  time,  this  collects 
in  a  or  b,  and  a  corresponding  amount  of  water  is  driven  into  the 
ascension  pipe  c.  The  height  of  the  water  in  the  ascension  pipe 
is  noted,  the  vessel  a  is  opened,  and  the  reaction  of  it  is  tested 
with  litmus  paper,  and  compared  with  the  reaction  before  the 
test  was  instituted. 

Normally,  very  little  or  no  gas  is  found,  and  the  original  re- 
action of  the  feces  does  not  change  materially.  If  c  is  filled  one- 
half  or  more,  pathological  conditions  are  indicated.  If  at  the 
same  time,  the  reaction  has  become  decidedly  more  acid,  car- 
bohydrate fermentation  has  taken  place  (the  positive  result  of 

1.  See  Fig.  13,  Appendix. 


ANALYSIS  OF  FECES  23 

the  fermentation  test),  which  is  further  indicated  by  the  light 
color  of  the  feces.  If  the  reaction  has  become  more  alkaline 
and  the  color  is  dark,  putrefaction  is  indicated.  Both  fermenta- 
tion and  putrefaction  may  occur  at  the  same  time,  in  which  case 
the  reaction  may  be  neutral. 

6.  Dissolved  Albumin.— From.  20  to  30  grams  of  the  feces  are 
ground  up  with  water,  which  should  be  added  gradually  in  small 
quantities,  until  thinned  into  a  fluid  consistency.  It  is  allowed 
to  stand  for  several  hours  and  then  filtered  through  a  double 
filter.  The  muddy  filtrate  is  clarified  with  some  pure  diatom- 
aceous  earth.  To  this  clear  filtrate,  36  per  cent,  acetic  acid  is 
added,  drop  by  drop,  as  long  as  a  turbidity  or  a  precipitate  is 
formed.  This  precipitate  is  the  {normal)  nucleoprotein  of  the 
feces.  The  material  is  again  filtered  with  the  aid  of  diatom- 
aceous  earth,  and  the  new  filtrate  is  tested  with  a  drop  of  potas- 
sium ferrocyanide  solution  to  ascertain  the  presence  of  dis- 
solved albumin   (and  albumose) . 

The  examination  for  dissolved  albumin  comes  into  considera- 
tion only  in  cases  of  diarrhea,  and  then  only  when  it  is  question- 
able whether  the  fundamental  disturbance  is  purely  functional 
or  organic.  It  is  a  matter  of  observation  that  a  diarrheal  stool, 
which  contains  no  mucus,  but  shows  a  reaction  for  albumin  and 
putrefies  in  the  incubator  test,  is  mixed  with  transudate  serum, 
and,  therefore,  points  to  an  irritated  or  inflammatory  condition, 
or  to  ulcers  of  the  mucous  membrane. 

7.  "  Occult  "  Blood. — The  test  for  blood  is  of  value  only  in 
cases  of  organic  disease,  and  then  it  has  for  its  condition  a  diet 
without  meat.  A  number  of  tests  for  occult  blood  have  been  sug- 
gested, among  such  being  the  guaiac,  benzidine  and  phenolphtha- 
lin  tests,  the  last  two  being  extremely  delicate  when  properly 
applied.  The  guaiac  test  is,  however,  sufficiently  delicate  for 
most  purposes. 

a.  Guaiac  Test. — About  5  to  10  grams  of  feces  are  stirred 
in  a  mortar  with  water  until  it  is  a  thick,  fluid-like  consis- 
tency. If  the  feces  contain  much  fat,  this  should  be  ex- 
tracted with  about  three  volumes  of  ether  and  poured  off. 
To  this  thick,  fluid-like  excrement  is  added  one-third  the 
volume  of  glacial  acetic  acid  and  thoroughly  mixed.  About 
10  cc.  of  the  entire  mass  are  poured  into  a  test  tube,  about 
the  same  amount  of  ether  added,  and  the  material   carefully, 


24  PATHOLOGICAL  CHEMISTRY 

but  thoroughly,  mixed.  The  ether  is  then  allowed  to  separate. 
This  may  be  quickly  accomplished  with  the  aid  ot  the  centri- 
fuge. In  the  presence  of  blood,  the  spectroscope  will  reveal 
absorption  bands  of  acid  hematin.  More  simply,  blood  may 
be  detected  in  this  ethereal  extract,  which  has  been  poured 
into  another  tube,  by  adding  10  drops  of  a  one  to  sixty  alcoholic 
solution  of  guaiac  resin,  followed  by  20  to  30  drops  of  ozonized 
turpentine,  or  about  four  cc.  of  10  per  cent,  hydrogen  peroxide. 
The  whole  is  shaken.  A  blue  coloring  shows  the  presence  of 
blood,  provided  that  there  is  no  pus  mixed  with  the  feces. 

b.  Benzidine  Test. — A  knife  point  full  of  pure  benzidine 
(must  be  kept  in  a  dark  place)  is  dissolved  in  two  to  three  cc. 
of  glacial  acetic  acid.  To  this,  are  added  two  cc.  of  hydrogen 
peroxide.  A  small  amount  of  feces  is  now  shaken  up  with 
a  little  hot  water,  and  about  three  cc.  of  the  suspension  added 
to  the  above  solution.  If  blood  is  present,  a  greenish  or  bluish 
reaction  will  occur  in  from  one  to  three  minutes.  Delicacy 
1-100,000. 

c.  Phenolphthalin  Test. — On  adding  about  one  cc.  of  the 
phenolphthalin  reagent1  to  a  solution  of  a  small  bit  of  the  sus- 
pected fecal  matter  in  water  (about  two  cc.)  and  treating  with 
one,  or,  at  most,  two  drops  of  a  10  per  cent,  solution  of  hydrogen 
peroxide,  a  bright  red  color  will  develop,  owing  to  a  reoxidation 
of  phenolphthalin  to  phenolphthalein  through  the  agency  of  the 
oxidase  of  the  blood  in  the  presence  of  the  peroxide.  Delicacy 
1-800,000. 

8.  Indentification  of  Gall-stones. — Gall-stones  are  occasionally 
found  in  the  feces  after  passage  from  the  gall-bladder,  but  only 
after  careful  washing  and  sifting  of  the  excreted  material.  Gall- 
stones are  classified  according  to  the  amount  of  the  various 
ingredients  which  they  contain.  In  man,  the  biliary  concretions 
are  generally  cholesterol  calculi,  or  pigment  calculi,  or  combina- 
tions of  the  two.  The  presence  of  these  constituents  serves  to 
identify  the  calculus  as  biliary  in  origin. 

1.  The  reagent  is  prepared  as  follows:  To  100  cc.  of  a  20  per  cent, 
solution  of  sodium  hydroxide  are  added  two  grams  of  phenolphthalein  and 
10  grams  of  zinc  dust.  The  bright  red  solution  is  heated  gradually  until 
it  has  become  decolorized,  or  rather  until  it  has  assumed  a  slight  yellowish 
tone,  owing  to  a  reduction  of  the  phenolphthalein  to  phenolphthalin. 
The  supernatant  fluid  is  poured  off  into  a  colored  glass  bottle,  and  the 
access  of  air  prevented  by  the  addition  of  a  little  liquid  paraffin  which 
floats  on  top. 


ANALYSIS  OF  DUODENAL  JUICE  25 

The  calculus  is  pulverized  in  a  clean  dry  mortar  and  extracted 
with  ether.  Upon  filtering  off  the  ether  and  allowing  it  to  evapo- 
rate, any  cholesterol  will  crystallize  out.  If  some  of  the  crystals 
are  dissolved  in  chloroform  and  stratified  upon  concentrated 
sulphuric  acid,  the  acid  will  take  on  a  greenish  fluorescence,  and 
the  chloroform  will  become  red.  The  residue  of  the  calculus, 
remaining  after  the  ether  extraction,  is  washed  with  dilute  hydro- 
chloric acid,  dried,  then  extracted  first  with  chloroform,  and 
afterwards  with  hot  alcohol.  The  chloroform  will  extract  the 
bilirubin,  and  the  alcohol  the  biliverdin,  the  former  imparting 
a  golden  yellow  color  to  the  solution,  the  latter  an  emerald  green. 

9 .  Examination  of  Duodenal  Juice. — Since  the  introduction 
of  the  Einhorn  duodenal  pump1-  making  it  possible  to  easily  ob- 
tain fluid  from  the  duodenum,  interest  has  arisen  in  this  fluid's 
being  made  of  diagnostic  value.  As  yet  the  examination  of  the 
duodenal  juice2  has  not  yielded  data  of  decided  value  in  diagno- 
sis, though  it  would  appear  to  be  the  most  logical  method  of  as- 
certaining a  deficiency  in  the  pancreatic  secretion.  Duodenal 
juice  obtained  in  this  way  is  normally  a  clear,  golden  yellow, 
slightly  viscid  fluid.  It  is  neutral  or  faintly  alkaline  in  reaction 
to  litmus  and  has  a  specific  gravity  of  about  1.005.  The  juice 
contains  bile,  as  its  color  would  indicate,  and  active  amylolytic, 
lipolytic  and  proteolytic  enzymes.  The  methods  which  we  have 
employed  for  estimating  the  enzyme  content  are  as  follows: 

a.  Amylase  (amylopsin) . — The  Wohlgemuth  method  is  simple 
and  fairly  satisfactory.  Into  each  of  six  small  test  tubes  are  in- 
troduced 5  cc.  of  1  per  cent,  soluable  starch  solution.  Tube  1 
serves  as  a  control  and  to  the  remaining  five  tubes  are  added  .05, 
.1,  .25,  .5  and  1.0  cc.  of  the  juice  diluted  one  half  with  distilled 
water.  The  tubes  are  then  incubated  at  38°  C  for  30  minutes, 
immediately  filled  nearly  full  with  cold  water,  several  drops  of 
N/10  iodine  added  and  the  tubes  shaken.  The  tube  is  selected 
as  positive  which  shows  an  entire  disappearance  of  all  blue  color. 
The  enzyme  activity  is  expressed  in  the  number  of  cc.  of  starch 
solution,  1  cc.  of  undiluted  juice  is  capable  of  digesting.  If  it 
takes  1  cc.  of  juice  to  digest  5  cc.  of  starch,  the  activity  is  5,  if 


1.  Cf.  Einhorn:  Diseases  of  the  Stomach,  New  York,  1911,    p.  86. 

2.  Cf.  Einhorn  and  Rosenbloom:  Arch  Int.  Med.,  1910,  VI,  p.  666; 
Hess;  Amer.  Jour.  Dis.  Child.,  1912,  IV.  p.  205  and  other  papers;  Crohn: 
Amer,  Jour.  Med.  Sci.  CXLV,  1913,  p.  393. 


26  PATHOLOGICAL  CHEMISTRY 

digestion  is  accomplished  by  .25  cc,  it  is  20,  etc.  For  the  five 
tubes  as  diluted  above  the  activity  figures  are  200,  100,  40,  20 
and  10.  The  activity  of  different  specimens  has  been  found 
to  vary  between  5  and  200,  although  the  average  figure  is  about 
40. 

b .  Lipase  (steapsin) . — Into  each  of  two  test  tubes  are 
introduced  1  cc.  portions  of  the  juice,  one  of  which  is  boiled 
to  serve  as  control.  To  each  of  these  tubes  are  added  1  cc.  of 
neutral  ethylbutyrate,  10  cc.  of  distilled  water  and  1  cc.  of 
toluene.  The  tubes  are  shaken  and  placed  in  the  incubator  at 
38°  C  for  24  hours,  shaking  several  times  during  the  interval. 
At  the  end  of  this  time,  they  are  removed  to  porcelain  dishes  and 
titrated  with  N/20  NaOH,  using  phenolphthalein  as  indicator. 
The  titration  result  of  the  boiled  tube  is  subtracted  from  the 
unboiled  to  obtain  the  figure  for  the  lipolytic  action.  We  have 
obtained  figures  varying  from  .3  -  4.3  cc.  The  average  has  been 
1.5-2.0. 

c.  Protease  (trypsin). — The  methods  available  for  estimating 
trypsin  are  not  as  satisfactory  as  those  for  pepsin.  Two  methods 
will  be  described,  the  Gross  casein  method,  and  a  modification 
of  the  Fermi  gelatin  method.  Casein  has  the  disadvantage  that 
it  is  also  attacked  by  erepsin,  while  the  gelatin  digestions  must 
be  carried  on  at  room  temperature. 

Casein  Method. — Into  each  of  six  small  test  tubes,  as  in  the 
amylase  method,  are  introduced  5  cc.  of  .1  per  cent,  pure  casein 
in  .1  per  cent,  sodium  carbonate1  and  the  same  amounts  of 
duodenal  juices  added  as  in  the  case  of  the  amylase.  The  tubes 
are  incubated  for  15  minutes  at  38°  C  and  then  acidified  with  a 
few  drops  of  dilute  acetic  acid.  The  tube  which  remains  per- 
fectly clear,  i.e.,  in  which  digestion  has  been  complete,  is  re- 
corded. The  tryptic  activity  may  be  calculated  in  the  same 
way  as  the  amylolytic  activity  above.  These  figures  are  exactly 
ten  times  those  obtained  according  to  the  original  Gross  calcu- 
lations, i.e.,  the  five  tubes  according  to  Gross  represent  an  activity 
of  20,  10,  4,  2  and  1.  By  this  method  we  have  found  the  activity 
ordinarily  to  be  4-10. 

Gelatin  Method. — Into  a  small  test  tube  is  pipetted  1  cc.  of 
undiluted  duodenal  juice  and  3  cc.  of  water,  1  cc.  of  .5  per  cent. 

1.  The  soluble  starch  and  casein  solutions  may  be  preserved  with 
chloroform  and  toluene  for  some  little  time,  especially  if  kept  in  a  re- 
frigerator. 


ANALYSIS  OF  DUODENAL  JUICE  27 

sodium  carbonate  and  1  cc.  of  toluene  added.  Into  the  test  tube 
is  then  inserted  two  3  cm.  gelatin  tubes1  and  the  tube  allowed  to 
incubate  for  48  hours  at  room  temperature  with  occasional  gentle 
mixing.  At  the  end  of  this  time  the  amount  of  digestion  is  mea- 
sured with  a  millimeter  scale  and  the  measurements  added  to- 
gether. The  average  total  digestion  with  this  method  in  some 
fifty  analyses  has  been  3.0  —  4.0  cm. 

1 .  The  gelatin  tubes  we  have  employed  have  been  prepared  by  dis- 
solving 10  grams  of  gelatin,  1  gram  of  sodium  fluoride  in  distilled  water, 
deeply  coloring  with  a  clear  solution  of  cochineal  and  making  up  to  100  cc. 
and  filling  tubes  of  2  mm.  inside  diameter.  The  tubes  3  cm.  in  length  are 
cut  just  previous  to  use. 


CHAPTER  III. 

The  Physical  Properties,  Inorganic  and  Organic 

Physiological  Constituents  of  Urine. 

Since  the  end  products  of  the  metabolism  of  nitrogenous  and 
mineral  substances  find  their  principal  exit  through  the  kidneys, 
a  study  of  the  secretion  of  these  glands  under  various  conditions 
may  be  expected  to  throw  light  upon  the  processes  involved  in 
the  metabolism  of  the  above  substances.  With  a  knowledge  of 
the  principal  constituents  of  the  urine  and  a  partial  under- 
standing, at  least,  of  their  history  in  the  body,  the  appearance 
of  any  unusual  substance  or  the  presence  of  a  normally  occurring 
constituent  in  an  amount  inconsistent  with  the  attending  con- 
ditions may  not  infrequently  serve  to  detect  derangements  of 
body  functions. 

The  mechanism  of  kidney  secretion  has  been  a  much  con- 
troverted question.  Perhaps  the  most  generally  held  view  at 
present  is  that  the  renal  epithelial  cells  actively  participate  in 
the  secretion,  the  water  and  inorganic  salts  being  eliminated  in 
the  capsular  region,  while  the  urea,  uric  acid,  etc.  find  their  exit 
through  the  uriniferous  tubules. 

Volume. — The  volume  of  urine  eliminated  depends  in  great 
part  upon  the  volume  of  fluid  ingested.  Under  normal  conditions 
one  liter  may  be  taken  as  the  average  volume  of  urine  excreted 
in  twenty-four  hours.  This,  however,  is  subject  to  great  vari- 
ations under  both  normal  and  pathological  conditions. 

The  secretory  activity  of  the  kidney  is  to  a  considerable 
extent  controlled  by  its  blood  supply,  the  latter,  in  turn,  being 
dependent  upon  general  blood  pressure  and  upon  the  state  of 
constriction  or  dilatation  of  the  renal  vessels.  Thus  where  the 
blood  pressure  is  raised,  as  for  example  in  chronic  nephritis, 
and  the  blood  supply  to  the  kidneys  is  consequently  augmented, 
one  observes  an  increased  secretion  of  urine.  Digitalis  may  ex- 
ert its  influence  in  this  manner,  although  it  probably  also  pro- 
duces a  stimulating  action  upon  the  secreting  cells  of  the  kidney. 
The  tendency  of  arterial  pressure  to  increase  the  blood  flow,  and 
consequently  to  augment  the  urinary  secretion,  may  be  masked 

28 


NORMAL  URINARY  CONSTITUENTS  29 

by  a  constriction  of  the  renal  vessels.  Such  a  condition  obtains 
in  strychnine  or  adrenaline  poisoning,  in  asphyxia,  eclampsia, 
etc.  The  flow  of  blood  is  increased  when  the  renal  vessels  are 
dilated,  although  this  may  be  counteracted  by  a  general  fall  in 
pressure,  in' which  case  there  would  be  no  actual  improvement 
in  the  renal  circulation.  Dilatation  of  the  vessels  of  the  kidney  is 
the  probable  explanation  for  the  large  volume  of  urine  eliminated 
in  diabetes  insipidus.  A  diminished  blood  flow  through  the 
kidneys  may  result  from  increased  venous  pressure — frequently 
associated  with  cardiac  diseases. 

The  condition  of  the  renal  epithelial  cells  influences  the  volume 
of  urine,  the  latter  being  usually  diminished  by  lesions  of  these 
cells.  However,  in  certain  cases  of  nephritis  the  blood  flow  is 
increased,  thus  tending  to  augment  the  secretion  of  urine.  It 
is  thus  apparent  that  in  nephritis  the  volume  of  the  secretion 
will  be  diminished  or  increased,  depending  upon  which  of  these 
factors  exerts  the  greater  influence.  Generally  in  acute  nephritis 
the  renal  changes  are  sufficiently  prominent  to  produce  decreased 
secretion,  while  in  chronic  nephritis  this  tendency  may  be  coun- 
teracted by  the  increased  arterial  pressure,  although  even  here 
when  cardiac  failure  ensues,  as  would  be  expected,  the  volume 
of  urine  eliminated  is  diminished. 

The  volume  of  urine  is  diminished  by  conditions  which  cause 
an  increased  elimination  of  water  through  other  channels,  for 
example  through  the  alimentary  tract  during  diarrhea  or  vomit- 
ing, or  through  the  skin  as  perspiration.  On  the  other  hand 
during  cold  weather,  when  cutaneous  evaporation  is  reduced, 
the  volume  of  urine  is  increased.  Thus  in  warm  weather  the 
volume  may  be  as  low  as  400  cc,  while  a  volume  of  2000  may  be 
encountered  during  cold  weather. 

When  the  kidneys  are  unable  to  properly  excrete  e.g.,  salt, 
giving  rise  to  edema,  water  is  retained  in  the  tissues  to  maintain 
normal  osmotic  relations.  On  the  contrary,  when  it  is  necessary 
to  eliminate  a  large  amount  of  material  as  is  the  case  with  sugar 
in  diabetes  mellitus,  the  volume  of  urine  is  increased. 

Opposition  to  the  flow  of  urine  may  be  encountered  at  any 
place  along  the  urinary  tract.  Such  obstruction  may  be  due  to 
scar  tissue,  calculi,  tumors,  etc.  If  but  one  kidney  or  ureter 
is  thus  affected,  the  total  urine  elimination  may  not  be  markedly 
influenced  as  the  other  kidney  will  very  likely  be  able  to  perform 
the  extra  work  thus  thrown  upon  it. 


30  PATHOLOGICAL  CHEMISTRY 

Color. — The  color  of  urine  may  vary  under  normal  con- 
ditions from  a  very  pale  yellow  to  a  reddish  yellow,  depending 
upon  its  density.  Pathologically  the  color  may  vary  from  a  light 
yellow  to  dark  brown  or  black.  A  red  color  may  be  due  to  blood ; 
very  dark  colored  urines  may  arise  after  taking  carbolic  acid; 
the  excretion  of  melanin  from  pigmented  tumors  may  likewise  be 
the  cause  of  a  dark  color,  especially  after  being  exposed  to  the 
air  for  some  time  or  on  the  addition  of  an  oxidizing  agent.  A 
green  or  brownish  yellow  color  may  be  due  to  bile.  Ingestion  of 
rhubarb,  senna  or  santonin  yields  urine  of  yellow  color,  which 
becomes  red  on  the  addition  of  an  alkali.  In  alkaptonuria — an 
anomaly  of  metabolism — the  urine  may  become  dark  owing  to 
the  presence  of  homogentisic  acid.  This  is  especially  so  if  the 
urine  is  allowed  to  become  alkaline.  Further  attention  is  de- 
voted to  this  topic  in  Chapter  VII. 

Specific  Gravity. — The  specific  gravity  of  normal  urine  most 
commonly  falls  between  1.015  and  1.025.  It  may,  however, 
be  as  low  as  1.010  or  as  high  as  1.040  without  necessarily  indica- 
ting pathological  conditions.  In  general,  both  normally  and 
pathologically,  the  specific  gravity  is  inversely  proportional  to 
the  volume.  In  diabetes  mellitus,  however,  we  may  observe  both 
a  large  volume  and  a  high  specific  gravity  owing  to  the  presence 
of  sugar. 

Reaction. — In  the  majority  of  cases  the  urine  is  acid  to 
litmus.  This  is  due  to  the  usual  preponderance  of  "acid 
radicals  "  over  M  basic  radicals."  The  acid  radicals  take  their 
origin  in  the  metabolism  of  proteins,  etc.,  during  which  are  pro- 
duced the  sulphuric  acid  and  a  part  of  the  phosphoric  acid  (from 
the  oxidation  of  the  sulphur  and  phosphorus  of  the  protein), 
and  organic  acids,  such  as  hippuric,  uric  and  oxalic  acids.  The 
'  basic  radicals  " — sodium,  potassium,  calcium,  magnesium  and 
ammonia — of  the  body  are  called  upon  to  partially  neutralize 
these  acids.  Ordinarily  the  latter  are  not  completely  neutral- 
ized, thus  accounting  for  the  customary  acid  nature  of  the  urine. 
The  reaction  of  the  urine  may  experience  marked  changes  under 
both  physiological  and  pathological  conditions. 

An  animal  dietary  yields  a  preponderance  of  acid-forming 
substances,  while  on  a  vegetable  diet  the  base  forming  elements 
are  usually  in  excess.1      Thus  an  highly  acid  urine  is  usually 

1.     Sherman  and  Gettler:     Jour.  Biol.  Chem.,  1912,  XI,  p.  323. 


NORMAL  URINARY  CONSTITUENTS  31 

associated  with  animal  food;  and  a  diet  containing  much  vege- 
table material  may  yield  neutral  or  even  alkaline  urine.  The 
foregoing  most  likely  accounts  for  the  fact  that  the  urine  of  dogs 
is  normally  acid,  while  that  of  rabbits  is  habitually  alkaline. 
That  this  difference  may  be  attributed  to  the  diet  is  shown  by  the 
fact  that  a  dog,  subsisting  on  a  vegetable  dietary  may  excrete  an 
alkaline  urine,  while  a  rabbit,  metabolizing  animal  material  (e.g., 
in  starvation)  may  eliminate  an  acid  urine. 

The  development  of  excessive  acidity,  owing  to  the  ingestion 
of  difficultly  oxidizable  acids  (mineral  acids) ,  or  the  pathological 
formation  of  acids  (as  in  diabetes),  is  counteracted  in  a  measure 
by  the  neutralizing  action  of  the  bases,  sodium,  potassium, 
calcium  and  magnesium.  When  the  acidity  is  so  great  that  an 
adequate  supply  of  these  elements  can  no  longer  be  economically 
furnished  by  the  body,  ammonia  is  called  upon  to  meet  this 
need.  This  accounts  for  the  relative  increase  of  ammonia  in 
severe  diabetes.  The  proximity  to  a  meal  may  affect  the  reac- 
tion of  the  urine.  For  example,  the  secretion  of  hydrochloric 
acid  into  the  stomach  during  the  process  of  digestion  may  so  re- 
duce the  store  of  acids  in  the  body,  that  for  a  time  after  a  meal, 
the  urine  may  be  neutral  or  even  alkaline,  giving  rise  to  the  so- 
called  "  alkaline  tide." 

Unless  certain  precautions  are  taken,  the  urine  sooner  or  later 
after  voiding,  becomes  alkaline  owing  to  the  conversion  of  urea 
into  ammonium  carbonate  by  bacteria.  In  cystitis  this  decom- 
position may  take  place  in  the  bladder.  If  a  urine  is  alkaline 
immediately  after  voiding,  one  should  decide  whether  the  alkalin- 
ity is  due  to  fixed  alkali  or  to  ammonia.  Only  in  the  latter  event 
is  cystitis  indicated.  An  alkaline  urine  may  be  the  result  of  ab- 
sorption and  excretion  of  alkaline  transudates. 

From  the  foregoing,  it  is  apparent  that  the  reaction  of  the 
urine  is  determined  by  the  character  of  the  diet,  proximity  to  a 
meal  and  presence  or  absence  of  ammonia-producing  organisms 
in  the  urine  either  before  or  after  voiding. 

Odor. — Normal  urine  has  a  characteristic  aromatic  odor.  The 
excretion  of  certain  drugs  (cubebs,  copaiba,  myrtol,  saffron, 
tolu  and  turpentine)  imparts  specific  odors  to  the  urine.  When 
the  latter  has  undergone  alkaline  fermentation,  a  disagreeable 
ammoniacal  odor  is  developed. 

Transparency. — When  voided  the  urine  of  a  normal  individual 


32  PATHOLOGICAL  CHEMISTRY 

is  usually  perfectly  clear.  On  standing  a  few  hours,  a  cloud  or 
"  nubecula  "  forms,  even  in  normal  urine.  This  cloud  consists  of 
mucus  threads,  epithelial  cells,  etc.,  from  the  urinary  passages. 
Under  pathological  conditions,  the  latter  may  be  greatly  in- 
creased and  accompanied  by  casts  or  blood.  If  the  acidity  of 
the  urine  is  somewhat  diminished  (as  after  a  meal)  a  turbidity 
due  to  phosphates  may  form.  This  will  disappear  on  adding  a 
little  acetic  acid.  On  standing  in  the  cold,  urates  may  settle  out 
but  will  again  go  into  solution  on  warming. 

Chlorides. — Under  ordinary  conditions  10  to  15  grams  of 
sodium  chloride  are  excreted  daily.  These  figures  are,  however, 
greatly  dependent  upon  the  salt  intake.  In  starvation  the 
sodium  chloride  excretion  is  reduced  to  a  minimum.  The  same 
conditions  obtain  in  cases  of  carcinoma  of  the  stomach,  resulting 
in  stenosis  of  the  pylorus,  essentially  a  condition  of  starvation. 
The  sodium  chloride  elimination  is  decreased  by  those  conditions 
which  favor  its  removal  from  the  blood  through  other  channels, 
e.g.,  cases  of  diarrhea,  rapidly  formed  transudates  and  exudates, 
such  as  pleurisy  with  effusion.  It  may  be  pointed  out  that  for 
several  days  after  the  reabsorption  of  an  exudate,  the  chloride 
excretion  may  be  greatly  increased,  and  is  here  a  favorable  diag- 
nostic sign.  Diminished  chloride  elimination  is  observed  during 
the  crises  of  acute  febrile  diseases,  especially  pneumonia  and 
in  chronic  nephritis,  in  the  latter  case  probably  because  of  the 
relative  impermeability  of  the  kidney  to  salts.  In  febrile  diseases 
it  is  worthy  of  note  that  the  elimination  of  chlorides  progressively 
decreases  as  the  febrile  process  approaches  its  crisis,  and  tends  to 
rise  to  its  original  level  during  convalescence.  The  chloride 
elimination  appears  to  be  augmented  by  exercise,  by  copious 
water  drinking,  and  in  diabetes  insipidus. 

Phosphates. — The  average  excretion  of  P2O5  is  one  to  five  grams 
daily.  This  originates  to  a  small  extent  from  the  oxidation  of 
the  phosphorus  of  protein  material.  It  owes  its  origin  in 
greater  part  to  the  phosphate  of  the  food,  and  the  extent  to 
which  the  latter  controls  the  phosphate  excretion  in  the  urine 
depends  upon  the  relative  abundance  of  alkali  and  alkali-earth 
phosphates.  The  alkali-earth  phosphates  are  difficultly  ab- 
sorbable and  hence  are  in  great  part  eliminated  directly  through 
the  feces,  thus  contributing  but  little  to  urinary  phosphates. 
The    alkali    phosphates   are    absorbed    and    add     to    urinarv 


NORMAL  URINARY  CONSTITUENTS  33 

phosphates  to  a  greater  extent,  but  even  these  may  be  con- 
verted into  alkali-earth  phosphates  in  the  body  and  be  in  part 
excreted  into  the  intestine,  reappearing  in  the  feces.  The 
phosphate  elimination  is  said  to  be  increased  in  periostosis, 
osteomalacia,  rickets  and  after  copious  water  drinking;  and 
decreased  in  acute  infectious  diseases,  pregnancy  and  diseases 
of  the  kidney.  At  times  a  turbidity  due  to  phosphates  is 
observed.  This  is  frequently  erroneously  interpreted  as  in- 
dicating an  increased  elimination  of  phosphates,  M  phosphaturia." 
It  is  more  likely  due  to  a  condition  of  decreased  acidity  and 
is  more  properly  termed  "  alkalmuria."  This  precipitation  of 
phosphates  may  also  be  due  to  an  unusual  amount  of  calcium 
which  would  form  one  of  the  less  soluble  phosphate  combinations. 

Sulphates. — Sulphur  is  excreted  in  three  forms:  oxidized  or 
inorganic  sulphur,  e.g.,  the  sulphates  of  sodium,  potassium, 
calcium  and  magnesium ;  ethereal  sulphur,  e.g., sulphates  of  phenol, 
indoxyl,  skatoxyl,  cresol,  etc.;  neutral  sulphur,  e.g.,  cystine, 
cysteine,  taurine,  hydrogen  sulphide,  etc.  The  greater  part  of 
the  sulphur  of  the  urine  is  present  in  the  oxidized  or  inorganic 
form,  averaging  about  2.5  grams  calculated  as  sulphuric  acid 
daily,  this  as  a  rule  being  about  ten  times  the  amount  of  ethereal 
sulphur  excreted.  The  inorganic  sulphur  of  the  urine  arises 
mainly  from  the  oxidation  of  the  sulphur  of  protein  material, 
and  is  thus  increased  by  those  conditions  which  stimulate  protein 
metabolism  as  acute  febrile  diseases  and  decreased  when  the 
rate  of  metabolism  is  lowered.  The  ethereal  sulphates  of  the 
urine  are  increased  by  excessive  formation  and  absorption  from 
the  intestine  of  products  of  putrefaction,  e.g.,  phenol,  indole, 
skatole,  or  by  the  administration  of  similar  aromatic  bodies 
such  as  phenol,  cresol,  resorcinol. 

Sodium,  Potassium,  Calcium  and  Magnesium. — The  quanti- 
ties of  these  elements  appearing  in  the  urine  are  subject  to  great 
variations  under  normal  conditions,  and  are  greatly  dependent 
upon  their  concentrations  in  the  food.  Very  little  is  known  re- 
garding their  pathological  variations.  Giving  approximate 
figures  it  may  be  said  that  the  urine  contains  daily  five  grams  sod- 
ium (as  Na20) ;  three  grams  potassium  (as  K20) ;  and  about  one 
gram  of  calcium  and  magnesium  phosphate  together.  Calcium 
salts  are  in  great  part  excreted  through  the  intestine,  which  con- 
dition makes  the  calcium  concentration  of  the  urine  an  unreliable 
index  to  the  extent  of  absorption  of  calcium  compounds. 


34  PATHOLOGICAL  CHEMISTRY 

Ammonia. — Under  ordinary  conditions  the  nitrogen  of  am- 
monia, in  combination  with  urinary  acids,  is  present  in  the  urine 
to  the  extent  of  2. 5 to 4. 5  per  cent,  of  the  total  nitrogen  eliminated, 
i.e.,  about  0.7  grams  per  day.  A  considerable  portion  of  this 
represents  ammonia  which  has  escaped  conversion  into  urea  so 
that  it  might  be  utilized  to  neutralize  the  sulphuric,  phosphoric, 
uric  acids,  etc.,  formed  in  the  process  of  normal  metabolism  or 
introduced  with  the  food.  This  procedure  probably  operates 
to  prevent  undue  drain  upon  the  body's  supply  of  sodium,  po- 
tassium, calcium  and  magnesium.  If  sufficient  fixed  alkalies  or 
alkali-earths  are  administered,  so  that  ammonia  is  not  required 
for  neutralizing  the  acids,  then  the  ammonia  excretion  may  be 
greatly  reduced,  or  in  fact  as  Janney  has  recently  shown1 
almost  completely  disappear  from  the  urine.  Furthermore,  as 
Sherman  and  Gettler2  have  demonstrated,  the  ammonia  output 
is  dependent  to  a  considerable  extent  upon  the  balance  between 
the  acid-forming  and  base-forming  elements  of  the  foods.  An 
injury  to  the  liver  cells  results  in  an  increased  output  of  am- 
monia owing  to  the  fact  that  these  cells  normally  convert  am- 
monium salts  to  urea.  Increased  elimination  of  ammonia  has 
been  observed  in  pernicious  vomiting  of  pregnancy.  It  is  im- 
portant to  note  that  here  the  individual  is  essentially  in  a  con- 
dition of  inanition,  which  itself  is  characterized  by  a  relative 
increase  in  ammonia  elimination.3 

A  very  large  number  of  organic  compounds  have  been  found 
in  normal  urine.  For  our  present  purposes  the  nitrogenous  sub- 
stances are  of  chief  interest  and  of  the  latter  urea,  uric  acid 
creatinine,  creatine,  hippuric  acid  and  the  purine  bases  will  be 
considered.  For  a  general  survey  of  the  state  of  protein  metabo- 
lism, the  determination  of  the  total  nitrogen  suffices,  and  indeed 
is  to  be  preferred  to  the  estimation  of  urea.  Although  the  latter 
represents  the  major  portion  of  the  total  nitrogen  (60  to  90  per 
cent.),  nevertheless  a  knowledge  of  the  amount  of  urea  alone 
leaves  one  in  ignorance  as  to  the  excretion  of  the  40-10  per 
cent,  of  nitrogen  in  other  combinations.  When  more  detailed 
or  specific  information  concerning  the  condition  of  nitrogenous 
metabolism  is  desired,  the  various  constituents  above  men- 
tioned may  be  determined  with  profit. 


1.  Janney:  Zeitschr.  f.  physiol.  Chem.,  1912,  LXXVI,  p.  99. 

2.  Sherman  and  Gettler;  Jour.  Biol.  Chem.,  1912,  XI,  p.  323. 

3.  Underhill  and  Rand:  Arch.  Int.  Med.,  1910,  V.,  p.  61. 


NORMAL  URINARY  CONSTITUENTS  35' 

Urea. — Urea  is  the  chief  end  product  of  protein  metabolism 
and  its  quantitative  excretion  is  closely  proportional  to  the 
amount  of  protein  ingested.  Thus  variations  of  10  to  40  grams 
are  not  uncommon.  The  percentage  of  urea  is  dependent  upon 
the  volume  of  urine  in  addition  to  the  protein  of  the  diet,  and 
when  it  is  considered  that  the  former  may  vary  from  500  cc.  to 
2000  cc,  it  is  evident  that  but  little  information  can  be  gained 
from  a  knowledge  of  merely  the  percentage  of  urea.  The  urea 
nitrogen  in  proportion  to  the  total  nitrogen  excreted  may  like- 
wise be  greatly  influenced  by  the  amount  of  protein  in  the  diet. 
Thus  with  a  high  protein  "intake,  the  urea  nitrogen  may  makeup 
as  much  as  90  per  cent,  of  the  total  nitrogen ;  while  with  a  diet  con- 
taining relatively  little  protein  but  considerable  carbohydrate  and 
fat,  the  proportion  may  be  as  low  as  60  per  cent.  With  a  nitrogen 
intake  of  20  grams  the  urine  would  contain  approximately  20 
grams  of  nitrogen  of  which  18  grams  may  be  in  the  form  of 
urea ;  whereas  with  a  nitrogen  intake  of  seven  grams  the  excretion 
of  urea  nitrogen  may  be  as  low  as  four  grams. 

The  origin  of  urea  may  be  considered  at  this  point.  It  is 
generally  held  that  the  amino  acids  formed  during  digesti  on  are 
carried  by  the  portal  circulation  to  the  liver  and  there  those  not 
needed  for  tissue  repair  (the  greater  part)  are  deamidized,  i.e., 
ammonia  is  split  off.  The  latter  uniting  with  the  carbonic  acid 
of  the  blood  forms  ammonium  carbonate,  which,  in  turn,  is 
converted  into  ammonium  carbamate  and  this  finally  into  urea. 
Precisely  the  manner  in  which  the  amino  acids  are  converted 
into  urea  is  still  a  matter  of  dispute.  Indeed  Folin  and  Denis 
have  recently  shown  that  the  amino  acids  are  not  deamidized  in 
the  intestinal  wall  or  immediately  in  the  liver,  but  may  be  carried 
unchanged  directly  to  all  parts  of  the  body.1  Nevertheless,  it 
may  be  assumed  that  ammonia  is  ultimately  split  off  and  is 
normally  converted  into  urea  principally  in  the  liver.  Con- 
sequently any  condition  which  hampers  the  liver  cells  from  trans- 
forming ammonia  into  urea  would  be  expected  to  lead  to  a 
diminished  output  of  the  latter  and  an  increased  elimination  of 
the  former.  Thus  in  cirrhosis  and  in  acute  yellow  atrophy, 
where  the  function  of  the  hepatic  cells  is  disturbed,  the  urea 
excretion  may  be  abnormally  low.  On  the  other  hand  it  must 
be  noted  that  even  in  extensive  alteration  in  the  liver,  the  relation 

1.  Folin  and  Denis:  Jour.  Biol.  Chem.,  1912,  XI,  p.  87;  ibid.  p.  161. 


36  PATHOLOGICAL  CHEMISTRY 

between  urea  and  ammonia  is  frequently  not  essentially  changed. 
A  diminished  urea  elimination  may  likewise  be  found  in  such 
conditions  as  acidosis  where  considerable  ammonia  is  utilized 
to  neutralize  acid  substances  before  it  can  be  transformed  into 
urea.  Impaired  kidneys  may  result  in  a  lowered  output  of 
urea  although  this  is  not  a  constant  association.  It  may  also 
be  noted  that  in  renal  disease  the  curve  for  the  urea  elimination 
discloses  eccentric  and  unaccountable  variations.  When  the 
rate  of  metabolism  is  accelerated  as  in  fevers,  exopthalmic 
goitre,  etc.,  the  total  nitrogen  and  urea  are  augmented. 

As  already  pointed  out,  it  is  quite  essential  in  considering  the 
excretion  of  total  nitrogen  and  urea  to  compare  these  values  with 
the  nitrogen  of  the  food,  because,  only  when  the  nitrogen  out- 
put is  out  of  proportion  to  the  intake  can  an  abnormal  con- 
dition be  presumed  to  exist. 

Uric  Acid. — Uric  acid  results  from  the  cleavage  and  oxidation 
of  nuclear  material.  Nucleoprotein  is  split  into  protein  and 
nucleic  acid.  When  the  nucleoprotein  is  present  in  the  food,  this 
process  takes  place  in  the  alimentary  tract  under  the  influence 
of  trypsin;  when  the  body  cells  are  the  source  of  the  nucleopro- 
tein this  transformation  takes  place  in  the  tissues  probably 
through  the  agency  of  a  similar  enzyme.  In  either  case,  the 
nucleic  acid  yields  the  purine  bases,  adenine  and  guanine,  through 
the  agency  of  the  enzyme  nuclease.  Adenine  and  guanine  are 
then  converted  respectively  into  hypoxanthine  and  xanthine, 
this  change  being  accomplished  by  the  enzymes  adenase  and 
guanase.  Finally  by  means  of  an  oxidizing  enzyme,  xanthine  is 
transformed  into  uric  acid.  This  process' may  be  represented  as 
follows '} 

Nucleoprotein 

!        ! 

Nucleic  acid  Protein 

!      I 

Adenine     Guanine 

!  I 

Hypoxanthine  *-$>  Xanthine  ■»-*•  Uric  acid 


1.  For  a  more   detailed   consideration  of   these   transformations    see 
Myers;  Albany  Med.  Ann.,  1911,  XXXII,  p.  645. 


NORMAL  URINARY  CONSTITUENTS  37 

It  has  been  claimed  that  in  man  about  half  the  uric  acid  is 
further  subjected  to  an  enzymatic  change  (uricolysis) ,  being 
partially  converted  to  urea.  This,  however,  is  still  a  disputed 
question,  although  it  undoubtedly  takes  place  in  dogs.  A  small 
amount  of  the  above  mentioned  adenine,  guanine,  etc.  escapes 
conversion  into  uric  acid,  and  thus  gives  rise  to  the  purine  bases 
of   the    urine. 

The  precursors  of  uric  acid — nucleoprotein  and  purine  bases — 
may  be  present  in  the  food  or  in  the  disintegrating  cellular  matter 
of  the  body.  In  the  former  case  the  uric  acid  is  said  to  be  of 
"  exogenous  origin;"  in  the  latter  of  "  endogenous  origin." 
For  practical  purposes  it  may  be  stated  that  "  endogenous  " 
uric  acid  varies  among  individuals  from  0.2  gram  to  0.5  gram 
being  fairly  constant  for  a  given  individual  and  essentially  in- 
dependent of  the  the  protein  intake.  Any  uric  acid  in  excess 
of  this  is  to  be  attributed  to  substances  of  the  food,  which  in 
the  process  of  digestion  and  assimilation  yield  uric  acid.  Such 
food  materials  are  meat,  meat  extracts,  pancreas,  liver,  thymus, 
etc.,  also  vegetable  seed  materials,  e.g.,  peas  and  beans. 
On  a  mixed  diet  0.7  gram  of  uric  acid  may  be  taken  as  an 
average. 

Ordinarily  uric  acid  is  present  in  the  urine  as  sodium,  potassium 
or  ammonium  urate.  Only  when  the  urine  is  especially  acid 
does  uric  acid  itself  separate  out.  When  the  urine  is  con- 
centrated or  after  the  ingestion  of  considerable  meat,  pan- 
creas, etc.,  urates  may  be  deposited  shortly  after  the  urine 
is  voided.  In  other  cases  such  deposits  may  form  on  standing 
in  a  cool  place.     These  urate  sediments  dissolve  on  warming. 

The  greatest  increase  in  uric  acid  elimination  is  observed  in 
leukemia,  as  much  as  12  grams  having  been  found  to  be  excreted 
in  the  24  hours.  This  high  elimination  of  uric  acid  is  without 
doubt  to  be  referred  to  the  enormous  increase  in  the  number  of 
leucocytes  and  consequent  leukolysis.  An  increased  uric  acid 
excretion  is  observed  in  other  diseases  associated  with  a  high 
grade  of  leucocytosis. 

Our  knowledge  of  the  relation  of  uric  acid  to  gout  is  at  present 
in  an  exceedingly  unsettled  condition  and  the  question  cannot 
be  fully  discussed  here.  We  may  say  briefly  that  the  quantitative 
excretion  of  uric  acid  in  gouty  individuals  does  not  differ  markedly 
from  that  found  normally.     It  may,  however,  be  noted  that  for 


38  PATHOLOGICAL  CHEMISTRY 

two  or  three  days  preceding  an  attack  of  acute  gout  the  uric 
acid  elimination  is  diminished;  while  during  and  for  a  few  days 
after  the  attack  it  may  maintain  a  level  somewhat  above 
normal. 

The  lithia  and  piperazine  therapy  has  been  employed  with  the 
object  of  presenting  media  in  which  uric  acid  and  urates  are  more 
soluble  than  they  are  in  blood  or  urine.  However,  it  is  unlikely 
that  these  substances  will  react  with  the  uric  acid  any  more  read- 
ily than  with  the  other  acids  of  the  body;  and  hence  the  portion  of 
these  therapeutic  agents  actually  combining  with  the  uric  acid 
is  probably  insignificant.  "  Atophan  "  does  indeed  frequently 
increase  the  elimination  of  uric  acid  and  is  said  to  be  helpful 
in  acute  gout.  Nevertheless,  whether  or  not  this  drug  is  of 
lasting  benefit  is  an  open  question.  It  has  been  pointed  out, 
for  example,  that  atophan  produces  only  a  temporary  increase  of 
uric  acid  output,  which  is  compensated  by  a  subsequently  lowered 
elimination.1  ,  This  suggests,  according  to  Dohrn,  that  atophan 
produces  its  initial  effect  by  stimulating  the  formation  of  uric 
acid  from  its  immediate  precursors,  and,  when  the  store  of  the 
latter  is  exhausted,  no  further  increase  in  the  elimination  of  uric 
acid  takes  place — in  fact  a  lowered  output  may  result. 

Creatinine. — Our  accurate  knowledge  with  regard  to  the 
elimination  of  creatinine  is  of  very  recent  date,  namely,  since 
the  introduction  of  the  Folin  colorimetric  method  for  its  estima- 
tion in  1904.  During  this  interval,  creatinine  has  perhaps  re- 
ceived greater  attention  at  the  hands  of  investigators  than  any 
of  the  other  nitrogenous  urinary  constituents.2  Creatinine 
is  very  probably  derived  from  the  creatine  of  muscle  or  some 
common  precursor  substance,  but  just  where  or  how  this  trans- 
formation takes  place  has  not  been  ascertained.  The  quantity 
of  creatinine  eliminated  is  independent  of  either  the  amount  of 
protein  in  the  food  or  of  the  total  nitrogen  in  the  urine  and  is 
almost  absolutely  constant  from  hour  to  hour  and  from  day  to 
day  for  a  given  normal  individual.  This  statement  refers  strictly 
speaking  to  an  individual  upon  a  creatinine  and  creatine  free 
diet,  though  ordinarily  the  urinary  creatinine  is  almost  entirely 
endogenous    in    origin.     Ingested    creatinine    quite    largely   re- 


1.  Cf.  Dohrn:  Zeitschr.  f.  klin.  Med.,  1912,  LXXIV,  p.  445. 

2.  For  reveiw  of  literature  see  Myers:  Amer.  Jour.  Med.    Set.,    1910 
CXXXIX,  p.  256. 


NORMAL  URINARY  CONSTITUENTS  39 

appears  in  the  urine,  but  ingested  creatine  influences  the  creat- 
inine elimination  only  slightly,  if  at  all,  and  does  not  to  any 
extent  pass  into  the  urine  unchanged.  This  latter  fact,  first 
observed  by  Folin,  has  thrown  a  degree  of  doubt  on  the  con- 
nection of  these  two  bodies  in  metabolism.  Neither  decreased  nor 
increased  muscular  activity  uncomplicated  by  other  factors 
has  any  effect  upon  the  creatinine  elimination.  While  the  daily 
creatinine  excretion  is  practically  constant  for  each  healthy  in- 
dividual, different  persons  excrete  different  amounts  and  Folin 
first  noted  that  the  chief  factor  determining  this  was  the  weight 
of  the  person.  He  further  observed  that  the  fatter  the  subject 
the  less  creatinine  was  excreted  per  kilo  of  body  weight,  and  con- 
cluded from  this  that  the  amount  of  the  creatinine  excretion  de- 
pended primarily  upon  the  mass  of  active  protoplasmic  tissue. 
Benedict  and  Myers1  found  that  the  creatinine  excreted  by 
women  was,  in  general  much  lower  than  in  the  case  of  men, 
doubtless  due  to  their  poorer  muscular  development.  Creatinine 
is  by  far  the  most  reliable  index  as  to  the  amount  of  a  certain 
kind  of  true  tissue  katabolism  occurring  daily  in  any  given  in- 
dividual, and  further  appears  to  be  an  index  of  some  special  pro- 
cess of  "normal  metabolism  taking  place  largely  if  not  entirely  in 
the  muscles.  The  intensity  of  this  process  appears  to  be  asso- 
ciated with  the  muscular  strength  of  the  individual.  Normally 
one  to  two  grams  of  creatinine  are  eliminated  daily.  It  has  been 
found  convenient  to  express  this  in  milligrams  of  creatinine 
nitrogen  per  kilo  of  body  weight,  and  this  "creatinine  coefficient  " 
varies  between  7  and  11  for  a  strictly  normal  individual. 

A  low  creatinine  elimination  has  been  found  to  be  associated 
with  a  large  number  of  pathological  conditions,  especially  those 
accompanied  by  muscular  weakness,  such  as  exophthalmic 
goitre,  muscular  dystrophy,  carcinoma  of  the  liver,  etc.  Benedict 
and  Myers  observed  a  creatinine  coefficient  as  low  as  2  in  two 
very  old,  decrepit  women.  To  a  certain  extent,  the  creatinine 
elimination  appears  to  serve  as  an  index  to  the  physical  condition 
of  fitness  of  a  given  individual.  In  diseases  where  the  creatinine 
output  is  lowered,  creatine  is  generally  excreted. 

An  increased  creatinine  elimination  is  observed  in  fevers,  the 
rise  in  the  excretion  paralleling  the  rise  in  body  temperature 
very  closely,  likewise  the  elimination  of  total  nitrogen.     The  in- 

1.  Benedict  and  Myers:  Amer.  Jour.  Physiol.,  1907,  XVIII,  p.  377. 


40  PATHOLOGICAL  CHEMISTRY 

creased  excretion  is  entirely  due  to  the  hyperthermia,  and  crea- 
tinine is  thus  an  index  of  the  amount  of  the  increased  metabolism 
due  solely  to  this  cause,  as  pointed  out  by  Myers  and  Volovic.1 
They  have  found  in  experimental  fevers  in  rabbits,  that  the 
elimination  is  ordinarily  increased  about  35  per  cent.  Creatinine 
is  still  an  index  of  the  amount  of  a  certain  kind  of  endogenous 
metabolism,  which  is  proceeding  here  at  an  abnormal  intensity. 
Creatine. — Creatine  is  a  constant  constituent  of  both  striated 
and  non-striated  muscle,  though  it  exists  in  much  smaller  quan- 
tities in  the  former.  The  creatine  concentration  of  striated 
muscle  appears  to  be  both  constant  and  distinctive  for  a  given  spe- 
cies.2 Creatine  does  not  appear  to  be  a  normal  constituent  of  adult 
urine,  though  the  recent  results  of  Rose3  have  shown  its  pres- 
ence in  the  urine  during  infancy  and  childhood.  Benedict4  was 
the  first  worker  to  note  the  appearance  of  creatine  in  the  urine  in 
considerable  quantity,  and  that  during  inanition,  an  observation 
which  has  been  abundantly  confirmed.  Subsequently  Benedict 
and  Myers5  reported  the  elimination  of  creatine  in  a  considerable 
number  of  pathological  conditions,  in  which  its  appearance  was  to 
be  accounted  for  by  the  poor  nutritive  condition  of  the  patients. 
The  elimination  of  creatine  has  since  been  noted  in  a  variety  of 
diseases,  among  which  are  exophthalmic  goitre,  in  convalescence 
after  typhoid  fever,  muscular  dystrophy,  anterior  poliomyelitis, 
pernicious  vomiting  of  pregnancy,  carcinoma  of  the  liver,  etc. 
In  the  last  mentioned  disease  the  amount  eliminated  is  very  large, 
1  -1.5  grams.  In  many  of  these  cases,  notably  fevers  and  hepatic 
carcinoma,  under-nutrition  is  an  important  factor  in  the  produc- 
tion of  creatine.  The  appearance  of  creatine  is  generally  found 
to  be  associated  with  a  loss  of  muscle  protein,  thus  indicating 
its  source  to  be  the  creatine  of  muscle  tissue.  Recent  ex- 
periments,6 in  which  the  decrease  in  the  creatine  store  ot  the 
body  during  starvation  has  been  accounted  tor  by  the  amount  of 
its  excretion  in  the  urine ,  appear  to  demonstrate  this  origin .  When 
the  muscle  protein  is  used  to  supply  abnormal  demands  made  upon 
it  (energy),  the  creatine  of  the  urine  is  possibly  an  index  of  the 

1.  Myers  and  Volovic:  Jour.  Biol.  Chem.,  1913,  XIV,  p.  489. 

2.  Myers  and  Fine:  ibid.,  1913,  XIV,  p.  9. 

3.  Rose:  ibid.,  1911,  X,  p.  265. 

4.  Benedict:  Carnegie  Inst.  Wash.,  1907,  Pub.  No.  77,  p.  386. 

5.  Benedict  and, Myers:  Amer.  Jour.  Physiol.,  1907,  XVIII,  p.  407. 

6.  Myers  and  Fine:     Proc.  Soc.  Exp.  Biol.  Med.,  1912,  X,  p.  12. 


QUANTITATIVE  ANALYSIS  OF  URINE  41 

amount  of  this  protein  destruction.  Substances  which  exert  a 
sparing  effect  on  protein  metabolism,  such  as  carbohydrates, 
might  be  expected  to  decrease  or  prevent  the  elimination  of  creat- 
ine. That  carbohydrate  alone  will  cause  the  creatine  of  the 
urine  in  starvation  to  disappear  has  been  demonstrated. 

Hippuric  Acid. — On  a  mixed  diet  an  average  of  0.7  gram  of 
hippuric  acid  is  eliminated  in  the  urine.  Hippuric  acid  is  a  com- 
bination of  glycocoll  and  benzoic  acid.  The  former  arises 
from  the  protein  substances  of  the  body ;  while  the  latter  owes  its 
origin  in  part  to  certain  aromatic  bodies  of  the  food  (vegetable) 
which  are  ultimately  converted  into  benzoic  acid,  and  in  part  to 
putrefactive  changes  in  the  intestine.  Ingestion  of  benzoic  acid 
itself  or  its  salts  increase  the  output  of  hippuric  acid. 

Oxalic  Acid. — Oxalic  acid  in  the  form  of  calcium  oxalate 
usually  occurs  in  the  urine  in  very  small  amounts,  about  0.02 
gram  in  24  hours.  Oxalic  acid  is  probably  formed  from  the  metab- 
olism of  proteins  and  fat.  Its  output  may  be  increased  by  in- 
gesting foods  which  contain  oxalic  acid.  Such  foods  are  cabbage, 
spinach,    apples,    grapes,    etc. 

LABORATORY    PROCEDURES. 

When  the  examination  is  to  be  a  quantitative  one,  it  is  im- 
perative to  collect  a  twenty-four  hour  sample,  to  which  a  pre- 
servative is  added,  such  as  toluene,  thymol  or  chloroform.  The 
color  and  specific  gravity  taken  together  with  the  volume  are 
valuable  as  means  of  orientation,  and  may  at  times  serve  to  in- 
dicate some  unusual  condition.  Thus  a  small  volume  of  highly 
colored  urine  usually  has  a  relatively  high  specific  gravity ;  while 
a  low  specific  gravity  is  commonly  associated  with  a  large  volume 
of  pale  urine.  Occasionally  a  light  colored  urine  with  a  high 
specific  gravity  is  obtained — the  characteristic  type  observed  in 
diabetes.  The  reaction  is  noted  by  means  of  red  and  blue  litmus 
paper.  If  alkaline,  the  nature  of  the  alkalinity  should  be  deter- 
mined— whether  due  to  fixed  or  volatile  alkali.  The  latter  con- 
dition may  be  accompanied  by  an  ammoniacal  odor.  When 
the  urine  is  not  transparent,  the  turbidity  or  sediment  is  usually 
due  to  (a)  urates,  (b)  phosphates,  (c)  bacteria,  pus,  epithelial  cells, 
etc.  If  urates,  the  sediment  will  disappear  on  warming;  phos- 
phates dissolve  on  the  addition  of  dilute  acetic  acid;  while  the 
turbidity  due  to  the  formed  elements  will  not  be  affected  under 
this  treatment. 


42  PATHOLOGICAL  CHEMISTRY 

1.  Volume. — This  is  conveniently  measured  in  a  one  or  two 
liter  graduated  cylinder. 

2.  Color. — The  color  is  recorded  following  some  such  color 
scheme  as  that  of  the  Vogel  scale  in  which  the  colors  are  pale  yel- 
low, light  yellow,  yellow,  reddish  yellow  (amber),  yellowish  red 
(deep  amber),  red,  brownish  red,  reddish  brown,  brownish  black, 
black. 

3.  Specific  Gravity. — This  is  ordinarily  ascertained  with  the 
aid  of  the  urinometer.  A  large  test  tube,  or  the  cylinder  usually 
accompanying  the  urinometer  is  three-fourths  filled  with  urine. 
The  urinometer  is  then  allowed  to  sink  in  the  fluid  and  the  read- 
ing taken  from  the  lower  portion  of  the  meniscus  in  contact  with 
the  stem  of  the  instrument.  If  the  observation  is  not  made  at  the 
temperature  for  which  the  instrument  is  calibrated,  a  correction 
should  be  made  when  an  accurate  observation  is  necessary  as  in 
the  calculation  of  total  solids  or  in  the  estimation  of  sugar  by 
the  specific  gravity  method.  Most  instruments  are  graduated 
to  be  read  at  15°C.  The  correction  for  temperature  is  roughly 
made,  by  adding  0.001  for  every  3°C  above  this  15°C  and  the 
same  subtracted  for  every  3°C  below. 

4.  Total  Solids. — The  quantity  of  the  total  solids  eliminated 
in  the "  twenty-four  hours  may  be  approximately  computed  by 
multiplying  the  second  and  third  decimal  figures  of  the  specific 
gravity  by  2.6.  This  gives  the  number  of  grams  of  solids  in  one 
liter  of  urine.     From  this  the  24  hour  amount  may  be  calculated. 

5.  Chlorides. — (Volhard-Harvey  Method)1.  Five  cubic  cen- 
timeters of  urine  are  pipetted  into  a  small  porcelain  evaporating 
dish  and  diluted  with  about  20  cc.  of  distilled  water.  The  chlo- 
rides are  now  precipitated  with  exactly  10  cc.  of  the  standard 
silver  nitrate  solution2  and  about  two  cc.  of  the  indicator3  added. 
Standard  ammonium  sulphocyanate  solution4  is  then  run  in  from 

1.  Harvey:  Arch.  Int.  Med.  1910,  VI,  p.   12. 

2.  29.06  grams  of  silver  nitrate  dissolved  in  and  made  up  to  one 
liter  with  distilled  water.  Each-  cubic  centimeter  of  such  a  solution  is 
equivalent  to  0.01  gram  of  sodium  chloride. 

3.  100  grams  of  crystalline  ferric  ammonium  sulphate  dissolved  in 
100  cc.  of  25  per  cent,  nitric  acid. 

4.  Dissolve  about  13  grams  of  ammonium  sulphocyanate  in  800  cc. 
of  distilled  water.  According  to  the  method  described  in  the  text,  titrate 
this  solution  against  the  standard  silver  nitrate  and  estimate  how  much 
water  should  be  added  to  the  remainder  of  the  sulphocyanate  solution  to 
make  it  exactly  equivalent  to  the  standard  silver  solution. 


QUANTITATIVE  ANALYSIS  OF  URINE  43 

a  burette  until  the  first  trace  of  yellow  shows  throughout  the 
mixture.  By  subtracting  the  number  of  cubic  centimeters 
of  sulphocyanate  thus  employed  from  ten  and  multiplying  by 
0.01,  the  number  of  grams  of  sodium  chloride  in  five  cc.  of  urine  are 
obtained.  From  this  the  total  chloride  output  for  the  twenty- 
four  hours  may  be  computed. 

6.  Phosphates. — To  50  cc.  of  urine  in  a  beaker  or  an  evaporating 
dish  are  added  five  cc.  of  accessory  solution1  and  the  mixture 
heated  to  the  boiling  point.  By  means  of  a  burette,  a  standard 
solution  of  uranium  nitrate2  is  then  run  into  the  hot  solution, 
until  a  drop  of  the  mixture  yields  a  brownish  coloration  when 
brought  in  contact  with  a  10  per  cent,  solution  of  potassium  ferro- 
cyanide  on  a  porcelain  plate.  The  number  of  grams  of  P2O5  in  50 
cc.  of  urine  is  estimated  by  multiplying  by  0.005  the  amount  of 
uranium  nitrate  solution  required,  and  from  this  the  quantity 
P2O5  in  the  total  volume  of  urine  can  be  readily  calculated. 

7.  Total  Acidity  and  Ammonia. — Both  these  determinations 
may  be  performed  on  the  same  sample — the  former  by  the  method 
of  Folin  and  the  latter  by  the  Ronchese-Malfatti  procedure.  To 
25  cc.  of  urine  in  a  250  cc.  flask  are  added  50  cc.  of  distilled  water, 
15  grams  of  powdered  potassium  oxalate  and  a  few  drops  of 
phenolphthalein.3  The  mixture  is  then  titrated  with  N/10 
NaOH4  until  a  permanent  pink  color  makes  its  appearance.  The 
total  acidity  is  expressed  in  terms  of  the  volume  of  standard 
alkali  necessary  to  neutralize  the  entire  twenty-four  specimen  of 
urine.  Five  cubic  centimeters  of  commercial  formalin,  which 
have  been  neutralized  to  phenolphthalein,  are  now  added,  and 
the  solution  again  titrated  with  the  standard  alkali  until  the 
previous  pink  color  is  again  obtained.  By  multiplying  the 
number  of  cubic  centimeters  thus  employed  by  0.0017,  the  grams 
of  ammonia  in  25  cc.  of  urine  are  learned;  and  from  this  the  total 
ammonia  output  for  the  twenty-four  hours  may  be  estimated. 

1.  100  grams  of  sodium  acetate  and  100  cc.  of  30  per  cent,  acetic  acid 
to  the  liter. 

2.  Dissolve  44.8  grams  of  uranium  nitrate  in  900  cc.  of  distilled  water. 
By  titrating  this  solution  with  a  standard  phosphate  solution  (14.7  grams 
HNaNH4P04.4H20  to  a  liter)  the  amount  of  water  to  be  added  to  the 
remainder  of  the  uranium  solution  so  that  one  cc.  will  be  equivalent  to  the 
0.005  grams  of  P2O5  can  be  calculated. 

3.  One  gram  of  phenolphthalein  dissolved  in  100  cc.  of  95  per  cent,  alcohol. 

4.  For  a  simple  method  ot  preparing  standard  acids  and  alkali  see 
footnote  Chapter  I,  p.  6. 


44  PATHOLOGICAL  CHEMISTRY 

If  the  ammonia  nitrogen  is  desired  0.0014  may  be  substituted  for 
0.0017. 

Ordinarily  the  above  mentioned  method  for  determining 
ammonia  gives  sufficiently  reliable  data.  However,  the  results 
are  slightly  high  owing  to  the  presence  of  amino  acids  and  for 
this  reason  when  more  accurate  data  are  required,  it  may  be  ad- 
visable to  employ  the  method  of  Folin. 

An  accurately  measured  volume  of  urine,  10  or  20  cc,  is  placed 
in  a  tall  cylinder  and  after  the  addition  of  about  one  gram  of 
sodium  carbonate  and  5  to  10  cc.  of  crude  petroleum,  the  ammonia 
is  aspirated  into  a  bottle  containing  a  definite  amount,  10  or  20 
cc,  of  N/10  sulphuric  acid  and  a  few  drops  of  congo  red1  as  indi- 
cator. When  the  larger  volume  of  urine  has  been  used,  it  is  ad- 
visable to  employ  the  Folin  absorption  tube.  In  either  case 
sufficient  distilled  water  must  be  added  to  completely  immerse 
the  absorption  tube  in  the  N/10  acid.  To  exclude  any  error 
from  ammonia  in  the  air,  the  air  current  is  first  made  to  pass 
through  dilute  sulphuric  acid.  For  20  cc.  of  urine,  about  one 
and  one-half  hours  will  transfer  the  ammonia  to  the  N/10  acid, 
while  with  10  cc,  one-half  hour  will  suffice.  Compressed  air  may 
be  employed  in  place  of  the  suction.  When  the  operation  is 
completed,  the  acid  not  used  is  titrated  with  N/10  alkali,  the. 
difference  being  due  to  the  ammonia  of  the  urine.  By  multiply- 
ing this  figure  by  0.0014,  the  grams  ammonia  nitrogen  in  the 
urine  employed  will  be  found  and  from  this  the  24  hour  elimina- 
tion may  be  calculated. 

8.   Total  Nitrogen2  (Kjeldahl  Method). — Place  5  cc.  of  urine  in  a 

1.  0.5  gram  of  congo  red  in  a  mixture  of  90  cc.  of  distilled  water  and 
10  cc.  of  95  per  cent,  alcohol. 

2.  Folin  and  his  coworkers  (Jour.  Biol.  Chem.,  1912,  XI,  p.  493  et 
seq.)  have  recently  suggested  microchemical  methods  for  the  esti- 
mation of  total  nitrogen,  urea  and  ammonia,  by  which  the  small 
amounts  of  nitrogen  obtained  are  ultimately  estimated  colorimet- 
rically  as  ammonia  by  means  of  Nessler's  reagent.  There  are 
certain  mechanical  and  chemical  difficulties  in  connection  with  these 
methods,  however,  which  render  them  less  available  in  the  examination 
of  urine  for  scientific  and  clinical  purposes,  than  the  older  methods  here 
described.  As  indicated  in  the  chapter  on  body  fluids,  Folin  and  Denis 
have  demonstrated  the  great  value  of  these  methods  in  the  examination 
of  blood,  etc.  where  the  delicacy  of  the  older  methods  is  insufficient. 

If  greater  simplicity  is  desired  for  urine,  the  methods  for  total  nitrogen 
and  urea  here  described,  may  be  modified  by  employing  1  or  2  cc.  of 
urine  and  smaller  pieces  of  apparatus. 


QUANTITATIVE  ANALYSIS  OF  URINE  45 

Kjeldahl  flask,  add  20  cc.  of  concentrated  sulphuric  acid  and 
a  spoonful  (2  grams)  of  potassium  sulphate  and  boil  the  mixture 
in  the  digestion  rack  until  it  is  entirely  colorless.  Allow  the 
flask  to  cool  and  dilute  the  contents  with  about  200  cc.  of  tap 
water.  Add  a  little  more  of  a  saturated  NaOH  solution  than  is 
necessary  to  neutralize  the  sulphuric  acid  (about  40  cc.)  By 
means  of  a  safety-tube  connect  the  flask  with  a  condenser  so 
arranged  that  the  delivery-tube  passes  into  a  vessel  containing  a 
known  volume  (50  cc.)  of  N/10  sulphuric  acid,  using  care  that 
the  end  of  the  deliverv  tube  reaches  beneath  the  surface  of  the 
fluid.  It  is  now  distilled  until  about  three-fourths  of  the  solution 
has  passed  over.  Titrate  the  unused  N/10  sulphuric  acid  by 
means  of  N/10  sodium  hydroxide  using  congo  red  as  indicator. 
One  cc.  of  N/10  sulphuric  acid  is  the  equivalent  of  0.0014  gram 
nitrogen.  After  ascertaining  the  amount  of  N/10  sulphuric  acid 
neutralized  by  the  distilled  ammonia,  calculate  the  amount  of 
nitrogen  in  5  cc.  of  urine  and  then  in  the  24  hour  specimen. 
9.   Urea.— 

a.  Benedict's  Method. — Five  cc.  of  urine  are  introduced  into 
a  rather  wide  test-tube,  about  three  grams  of  potassium  bisul- 
phate  and  one  gram  of  zinc  sulphate  added,  a  small  quantity 
of  powdered  pumice  and  a  bit  of  paraffin  are  introduced 
and  the  mixture  boiled  to  dryness  in  a  paraffin  bath  at  130°C  and 
finally  heated  for  one  hour  at  160-3°  C.  The  residue  is  then 
rinsed  into  a  Kjeldahl  flask  with  hot  water,  15-20  cc.  of  10  per 
cent.  NaOH  added  and  the  ammonia  is  distilled  as  in  the 
Kjeldahl  method.  Make  correction  for  the  amount  of  ammonia 
nitrogen  originally  present  in  the  urine  and  calculate  the  24  hour 
elimination  of  urea  nitrogen  and  of  urea.1 

b.  Hypobrornite  Method. — The  instrument  which  is  ordi- 
narily employed  in  the  clinical  laboratory  with  the  hypobrornite 
solution  is  the  Doremus  ureometer.  We  have  taken  occasion 
to  compare  the  results  obtained  with  several  of  these  instruments 
as  ordinarily  graduated,  with  figures  obtained  with  Benedict's 
method  and  also  with  figures  obtained  with  the  hypobro- 
rnite method  employing  a  gas  burette.  The  hypobrornite 
method  when  properly  carried  out  with  a  gas  burette  gives  values 
which  are  very  close  to  those  with  Benedict's  method  and  suffi- 


1.  The  amount  of  urea  excreted  may  be  estimated  by  multiplying  the 
urea-nitrogen  elimination  by  2.14. 


46  PATHOLOGICAL  CHEMISTRY 

ciently  accurate  for  clinical  purposes,  while  the  Doremus  instru- 
ments which  we  have  used  have  given  results  20-45  per  cent,  below 
the  actual  values.  Obviously  such  data  are  valueless.  A  Lunge 
or  Schiff  gas  burette  may  be  very  conveniently  employed  in 
measuring  the  amount  of  nitrogen  gas  evolved  by  the  hypobro- 
mite  solution,  but  where  these  are  not  available,  an  ordinary 
burette  may  be  used  by  inverting  and  partly  immersing  it  in 
water  in  a  tall  cylinder.  The  upper  end  of  the  burette  is  then 
connected  with  a  bottle  of  100  cc.  capacity  containing  about 
25  cc.  of  the  hypobromite  solution  and  a  small  test  tube  containing 
two  cc.  of  urine.  The  rubber  stopper  is  tightly  inserted  and  the 
burette  reading  recorded.  The  bottle  is  now  inclined  and  the 
urine  allowed  to  mix  with  the  hypobromite  solution.1  The  bottle 
is  shaken  at  intervals  for  several  minutes,  the  burette  is  raised  until 
the  water  level  is  the  same  both  inside  and  outside  the  burette 
and  the  reading  then  taken.  If  the  tables  of  Simon  and  Regnard2 
are  at  hand  the  value  in  grams  may  be  read  off  directly.  If  not 
the  following  formula  may  be  used : 

v  (p— T) 
w  - 


354.5X760  (1  +  0.003665  t) 

in  which  w  =  weight  of  urea  in  grams ;  v  =  observed  volume  of 
N  gas ;  p  =  barometric  pressure  in  mm.  of  mercury ;  T  =  tension  of 
aqueous  vapor  for  temperature  t;  and  t  =  temperature  C.  The 
values  of  the  T  for  the  ordinary  temperatures  are : 


Temperature 

Tension  in  mm. 

20°  C 

17.396 

21°  C 

18.505 

22°  C 

19.675 

23°  C 

20.909 

24°  C 

22.211 

25°  C 

23 . 582 

It  should  be  remembered  that  the  hypobromite  solution  de- 
composes not  only  urea,  but  also  any  free  ammonia  and  to  a  con- 

1.  The  solution  is  made  as  needed  by  mixing  one  part  each  of  two 
separate  solutions  with  three  parts  of  water.  Solution  (a)  contains  12.5 
grams  of  sodium  bromide  and  12.5  grams  of  bromine  in  100  cc.  of  water; 
and  solution  (b)  is  a  22.5  per  cent,  solution  of  sodium  hydroxide. 

2.  See  Wood:  Chemical  and  Microscopical  Diagnosis,  3rd.  Edit., 
1911,  p.  476. 


QUANTITATIVE  ANALYSIS  OF  URINE.  47 

siderable  extent  all  other  nitrogenous  substances.  With  a  low 
urea  and  a  high  ammonia  content  this  might  lead  to  a  very  con- 
siderable error. 

10 .  Uric  Acid  (Folin  Method)1. — Place  300  cc.  of  urine  in  a  tall 
cylinder  and  add  75  cc.  of  the  Folin-Shaffer  reagent.2  Filter, 
transfer  two  125  cc.  portions  (to  serve  as  duplicates)  to  two 
beakers,  add  five  cc.  of  concentrated  ammonium  hydroxide  and 
allow  the  mixture  to  stand  24  hours.  The  precipitated  ammonium 
urate  is  then  transferred  quantitatively  to  a  hard  filter  and  washed 
with  10  per  cent,  ammonium  sulphate  solution.  After  removing 
the  filter  paper  from  the  funnel  and  opening  it  up,  the  precipitate 
is  washed  with  about  100  cc.  of  water  back  into  the  same  beaker. 
To  this  15  cc.  of  concentrated  sulphuric  acid  are  added  and  the 
mixture  immediately  titrated  with  N/20  potassium  permangan- 
ate3 until  the  first  tinge  of  pink  color  extends  throughout  the 
fluid  after  the  addition  of  two  drops  of  the  permanganate  solution. 
The  average  of  these  two  titrations  multiplied  by  3.75  plus  a 
correction  of  3.0  mg.  for  the  solubility  of  the  ammonium  urate, 
gives  the  mgms.  of  uric  acid  in  100  cc.  of  urine.  From  this,  the 
calculation  for  the  24  hours  may  easily  be  made. 

11.  Creatinine  (Folin  Method). — Pour  a  little  N/2  potassium 
bichromate4  into  one  of  the  two  cylinders  of  the  colorimeter 
(Duboscq's)  and  carefully  adjust  the  depth  of  the  solution  to  the 
8  mm.  mark.  Place  10  cc.  of  urine  in  a  500  cc.  volumetric  flask, 
add  15  cc.  of  a  saturated  solution  of  picric  acid  and  five  cc.  of  a 
10  per  cent,  solution  of  sodium  hydroxide.  Shake  thoroughly  and 
allow  the  mixture  to  stand  for  five  minutes.  At  the  end  of  this 
interval  the  contents  of  the  500  cc.  flask  are  diluted  to  the  500  cc. 
mark  with  tap  water,  thoroughly  mixed  and  a  portion  poured  into 
the  empty  cylinder  of  the  colorimeter.      A  number  of  readings  are 

1.  Folin  and  Macallum  (Jour.  Biol.  Chem.,  1912,  XIII,  p.  363)  have 
recently  suggested  a  simple  colorimetric  method  for  the  determination  of 
uric  acid  in  urine.  For  several  reasons  the  method  here  described  is  to  be 
preferred  for  urine,  although,  on  account  of  its- delicacy,  this  new  method 
is  the  only  one  available  for  the  determination  of  uric  acid  in  the  blood. 
(See  Chapter  X.) 

2.  500  grams  of  ammonium  sulphate,  five  grams  uranium  acetate  and 
60  cc.  of  10  per  cent,  acetic  acid  in  650  cc.  of  distilled  water. 

3.  1.581  grams  of  potassium  permanganate  dissolved  in  one  liter  of 
distilled  water. 

4.  24.55  grams  to  the  liter. 


48  PATHOLOGICAL  CHEMISTRY 

taken  immediately.  8.1  divided  by  the  reading  obtained  multi- 
plied by  the  total  volume  of  urine  will  give  the  milligrams  of 
creatinine  in  the  24  hour  specimen. 

12.  Creatine  {Folin-Benedict  and  Myers  Method). — Pipette 
two  10  cc.  portions  of  the  urine  into  two  100  cc.  Elenmeyer  flasks, 
add  10  cc.  of  approximately  normal  hydrochloric  acid1  to  each  and 
heat  in  the  autoclave  at  twenty  pounds  for  one-half  hour.  This 
converts  any  creatine  to  creatinine.  At  the  end  of  the  interval 
remove  the  flasks  and  allow  them  to  cool.  Determine  the  creat- 
inine plus  creatine  as  above,  employing  in  this  case  10  cc.  of  the 
10  per  cent,  alkali  to  overcome  the  increased  acidity.  The 
difference  between  the  preformed  and  the  total  creatinine  gives 
creatine  in  terms  of  creatinine.  By  multiplying  this  value  by 
1.16  the  weight  of  the  creatine  may  be  obtained. 

1.  Prepared  by  diluting  100  cc.  of  concentrated  hydrochloric  acid  to 
one  liter  with  distilled  water. 


CHAPTER  IV. 
Albuminuria. 

The  presence  in  the  urine  of  protein  substances  may  con- 
veniently be  considered  under  the  heading  of  disorders  of 
protein  metabolism.  Practically  all  of  the  proteins  present 
in  the  body,  including  their  cleavage  products  have  been  found 
to  appear  in  the  urine  under  pathological  conditions.  Among 
these  are  serum  albumin  and  serum  globulin,  hemoglobin, 
fibrinogen,  nucleoprotein,  glucoprotein,  Bence  Jones'  protein, 
proteoses,  peptones  and  amino  acids,  notably  tyrosine,  leucine, 
cystine,  glycine,  etc.  As  previously  mentioned,  the  facts  at 
our  disposal  indicate  that  the  end  products  of  proteolytic 
digestion  are  the  amino  acids,  and  further,  that  they  are  absorbed 
into  the  blood  as  such.  Just  where  or  how  they  are  employed 
for  the  repair  of  body  tissue  or  for  the  formation  of  the  circu- 
lating proteins  of  the  blood,  has  not  been  ascertained.  The 
presence  of  a  protein  substance  in  urine  may  be  due  to  one  of 
several  factors,  to  the  increased  permeability  of  the  kidney,  to 
the  presence  in  the  blood  of  foreign  protein  material,  or  to  normal 
material  in  abnormal  amounts. 

Albumin.— In  referring  to  the  presence  of  albumin  in  the  urine 
clinically,  the  term  includes  both  the  blood  proteins,  serum 
globulin  as  well  as  serum  albumin,  though  the  latter  is  nearly 
always  in  excess.  The  ratio  which  exists  between  the  paraglob- 
ulin  and  the  albumin  of  the  blood  in  man  is  about  1  to  1.5, 
while  in  the  urine,  the  ratio  generally  falls  between  this  point 
and  1  to  2.3,  the  amount  of  the  globulin  being  the  variable 
factor.  In  contracted  kidney  and  in  chronic  passive  congestion, 
the  quotient  may  be  found  between  2 . 8  and  5.3,  though  in  cases 
of  nephritis  in  which  there  are  extensive  lesions  in  the  renal 
epithelium,  it  may  be  lowered,  and  in  amyloid  disease  it  may  be 
below  one.  From  the  foregoing,  it  is  apparent  that  albumin  and 
globulin  nearly  always  exist  together  in  the  urine,  though  the 
former  is,  as  a  rule,  the  more  abundant.  Cases  are  on  record, 
however,  where  very  large  amounts  of  globulin  have  been  elimi- 
nated in  the  urine. 

49 


50  PATHOLOGICAL  CHEMISTRY 

The  more  important  conditions  under  which  albumin  may 
appear  in  the  urine  may  be  referred  to  briefly  as  follows :  There 
are  persons,  apparently  perfectly  healthy,  who  continually, 
or  at  intervals,  secrete  urine  in  which  albumin  may  easily  be 
demonstrated.  The  kidneys  of  these  individuals  may  not 
necessarily  be  the  site  of  a  true  nephritis,  though  the  presence 
of  albumin  would  indicate  some  inefficiency. 

Under  the  head  of  the  so-called  physiological  albuminurias 
are  classed  those  cases,  which,  though  symptoms  may  be  absent, 
excrete  albumin  after  cold  baths,  violent  physical  exertion,  or 
following  the  taking  of  an  abundance  of  food,  especially  many 
raw  eggs.  In  this  latter  case  it  has  been  shown  that  a  portion 
of  the  albumin  may  be  unchanged  egg  albumin.  Recently, 
the  elimination  of  albumin  following  such  severe  athletic  con- 
tests as  basket  ball  has  been  shown  not  to  be  an  unusual  occur- 
rence.1 

Albuminuria  due  to  circulatory  disturbances  may  follow 
changes  in  the  kidney  resulting  from  the  altered  blood  pressure, 
which  is  frequently  observed  in  severe  and  uncompensated  heart 
lesions.  In  some  way,  not  clearly  understood,  the  retarded 
circulation  seems  to  injure  the  renal  cells.  The  quantity  of 
albumin  is  usually  small  and  a  few  hyaline  casts  may  be  observed. 

Injury  to  the  renal  cells  produced  by  toxic  substances,  whether 
mineral  or  organic  poisons,  may  result  in  albuminuria.  Among 
such  poisons  are  arsenic  and  uranium  compound,  chromates. 
cantharides,  ether,  etc. 

Albuminuria  occurs  in  many  of  the  well-known  febrile  con- 
ditions, i.e.,  typhoid  fever,  due  to  degenerative  changes  in  the 
kidney  epithelium,  possibly  produced  by  the  toxins  formed. 
In  the  severer  cases  this  may  develop  into  a  true  nephritis  with 
much  albumin  and  many  casts. 

The  albuminuria  of  nephritis2  is  of  particular  interest.  In 
acute  nephritis  the  elimination  of  large  amounts  of  albumin  is 
a  constant  and  most  important  symptom,  the  amount  eliminated 
being  in  general  in  proportion  to  the  severity  of  the  condition. 
Usually  about  five  to  eight  grams  of  albumin  are  eliminated  in 

1.  Fischer:     Nephritis,  New  York,  1912:  pp.  47  and  182. 

2.  Barker:  Amer.  Jour.  Med.  Sci.\  1913,  CXLV,  pp.  42-68,  has  re- 
cently given  a  most  interesting  and  comprehensive  discussion  of  the 
commoner  forms  of  renal  disease,  with  special  reference  to  the  knowledge 
of  them  most  useful  at  present  to  the  general  practitioner. 


ALBUMINURIA  51 

twenty-four  hours,  though  on  rare  occasions,  the  amount  may  reach 
20  grams.  The  elimination  of  albumin  is  likewise  a  constant 
association  of  chronic  parenchymatous  nephritis,  the  quantity 
eliminated  generally  amounting  to  five  grams  a  day,  though  it  may 
exceed  that  of  the  acute  variety  and  reach  to  15  to  30  grams 
daily.  In  the  majority  of  cases  of  chronic  interstitial  nephritis, 
on  the  other  hand,  the  elimination  of  albumin  is  slight,  rarely 
amounting  to  more  than  two  to  five  grams  per  day.  In  fact  it  is 
not  unusual  to  meet  with  an  apparent  absence  of  albumin,  as 
indicated  by  the  less  delicate  tests.  Obviously  a  careful  exam- 
ination for  albumin  and  casts  is  here  of  very  great  diagnostic 
importance.  The  amount  of  albumin  observed  in  amyloid 
degeneration  is  quite  comparable  to  chronic  interstitial  nephritis. 
An  entire  absence  of  albumin  is  less  frequent,  however,  the 
quantity  eliminated  generally  amounting  to  one  to  two  grams  per 
day,  though  as  pointed  out  above,  large  amounts  of  serum  globulin 
are  sometimes  excreted. 

Nucleo protein. — Nucleoprotein  occurs  in  normal  urine  only 
in  minute  traces,  but  in  larger  amounts  in  the  urine  of  the  new 
born,  after  over  exertion,  in  inflammation  of  the  mucous  mem- 
brane of  any  part  of  the  urinary  tract,  in  leukemia,  in  chronic 
parenchymatous  nephritis,  and  very  abundantly  during  jaundice. 
Clinically,  its  presence  in  the  urine  in  increased  amounts  is 
simply  an  indication  of  some  irritation  along  the  urinary  tract. 

Bence  Jones'  Protein. — In  association  with  multiple  myeloma 
of  the  bone,  a  peculiar  protein  substance  has  been  observed  in 
the  urine  in  80  per  cent,  of  the  cases  and  is  apparently  pathogno- 
monic of  the  disease.1 

Proteoses. — Proteoses  (albumoses)  are  present  in  traces  in 
the  urine  in  most  febrile  diseases  and  in  quite  a  variety  of  other 
conditions,  notably  those  in  which  a  septic  condition  exists  in 
some  part  of  the  body.  They  have  been  observed  in  ulceration 
of  the  intestine,  in  typhoid  and  in  dysentery,  occasionally  in 
malignant  growths,  probably  due  to  ulceration,  in  abscess  of 
the  liver,  in  empyema,  and  in  phosphorus  poisoning.  Their 
presence  in  the  urine  in  meningitis  is  supposed  to  be  in  favor 
of  a  suppurative  process,  and  against  a  tubercular  one. 

Amino  Acids. — Glycocoll  is  the  one  amino  acid  which  appears 
to  be  present!  n  normal  urine  in  small  amounts,  though  traces 

1.  For  literature  consult  Rosenbloom:     Biochem.  Bui.,  1911,  I,  p.  161. 


52  PATHOLOGICAL  CHEMISTRY 

of  other  amino  acids  probably  exist.  In  certain  severe  organic 
diseases  of  the  liver,  as  acute  yellow  atrophy,  chloroform  necrosis, 
phosphorus  poisoning,  eclampsia,  and  occasionally  in  severe 
infections  and  diabetic  coma,  different  amino  acids  appear  in 
the  urine,  leucine,  tyrosine,  and  cystine  being  the  most  important. 
Whether  the  presence  of  these  acids  in  the  urine  is  due  to  the 
inability  on  the  part  of  the  body  to  utilize  them  or  to  a  failure 
of  the  deaminization  reaction,  which  possibly  takes  place  in  the 
liver,  is  not  clear.  In  a  very  recent  paper,  Levene  and  Van 
Slyke1  report  a  series  of  determinations  of  the  amino  acid  con- 
tent of  normal  and  pathological  urines.  Cases  of  arithritis, 
gout,  carcinoma  of  the  breast  and  nephritis  were  observed  in 
which  there  was  a  notable  increase  in  the  elimination  of  nitro- 
gen in  this  form. 

In  two  very  interesting  but  rare  congenital  anomalies  of 
protein  metabolism,  cystinuria  and  alkaptonuria,  there  appears 
to  exist  a  faulty  metabolism  of  certain  individual  amino  acids. 
In  the  case  of  cystinuria,  the  sulphur  containing  amino  acid, 
cystine,  is  unutilized  and  is  eliminated  in  the  urine,  the  amount 
excreted  per  day  varying  between  a  few  centigrams  and  a  gram. 
In  the  case  of  alkaptonuria,  a  faulty  metabolism  of  the  phenyl 
amino  acids,  tyrosine  and  phenylalanine,  is  observed.  The  body 
appears  to  be  unable  to  carry  the  transformations  of  these  acids 
beyond  the  homogentisic'acid  (dioxyphenylacetic  acid)  stage 
and  this  compound  then  appears  in  the  urine.  Neither  of  these 
conditions,  however,  is  connected  with  any  clinical  symptoms. 
Metabolism  in  Renal  Disease  and  the  Permeability  of  the  Kidney. — 
Eccentric  and  unaccountable  variations  in  the  elimination  of 
the  urinary  nitrogen  are  observed  in  renal  diseases,  a  decreased 
elimination  being  followed  by  a  compensatory  increase.  In 
acute  nephritis,  however,  it  is  usual  to  find  a  decreased  elimina- 
tion of  nitrogen  and  especially  of  its  chief  component,  urea, 
though  there  are  periods  in  the  course  of  a  nephritis  in  which 
the  urea  excretion  is  perfectly  normal.  The  elimination  of 
ammonia  and  uric  acid  is  said  to  be  normal.  As  regards  the 
elimination  of  sodium  chloride,  its  retention  appears  to  go  hand 
in  hand  with  that  of  urea,  the  greatest  retention  being  likewise 
observed  in  acute  nephritis.  The  decreased  excretion  of  water  is 
coincident  with  the  formation  of  edema  and  the  retention  of 


1.   Levene  and  Van  Slyke:    Jour,  Biol.  Chem.,  1912,  XII,  p.  310. 


ALBUMINURIA  53 

salt,  in  which  the  sodium  chloride  appears  to  play  a  very  im- 
portant part.  Whether  the  salt  is  retained  because  of  the  imper- 
meability of  the  kidneys,  and  the  water  needed  to  preserve  the 
proper  osmotic  relations,  or  the  water  is  taken  up  by  the  hydro- 
phylic  colloids  of  the  tissues  and  the  salt  subsequently  retained 
to  maintain  the  normal  osmotic  pressure  of  the  body  fluids,  is 
not  clear. 

As  regards  the  nitrogen  intake  of  individuals  suffering  from 
nephritis,  it  seems  to  be  advisable  as  shown  by  von  Noorden1, 
to  reduce  the  nitrogen  intake  to  the  lowest  possible  level  in 
acute  nephritis,  as  here,  and  in  acute  inflammatory  exacerbations 
of  chronic  renal  disease,  a  large  protein  intake  undoubtedly 
exercises  an  injurious  effect  on  the  albuminuria.  Even  a  milk 
diet  is  too  rich.  In  the  chronic  forms,  the  protein  intake  should 
not  be  reduced  below  80  to  90  grams  per  day;  otherwise,  the 
patients  become  progressively  weaker.  Nitrogen  equilibrium  may 
be  maintained,  however,  even  with  considerable  loss  of  protein 
in  the  urine,  if  the  nitrogen  intake  is  high  enough.  The  extended 
observations  of  von  Noorden  would  indicate  that  in  interstitial 
nephritis — the  most  frequent  and  important  of  all  forms  of  kidney 
disease — the  degree  of  albuminuria  is  in  no  way  influenced  by 
the  protein  intake. 

Though  the  estimation  of  urea  has  long  been  employed 
clinically  as  a  method  of  estimating  the  efficiency  of  the  kidney, 
it  is  questionable  whether  it  is  ever  of  much  value  from  this 
standpoint,  even  when  the  urea  is  accurately  determined  and 
the  nitrogen  content  of  the  diet  known.  A  test,  which  has 
met  with  considerable  favor,  has  recently  been  described  by 
Rowntree  and  Geraghty  (see  below).  This  is  a  permeation  test 
in  which  a  known  amount  of  phenolsulphonephthalein  is  given, 
and  the  ability  of  the  kidney  to  eliminate  the  drug  ascertained. 
The  test  appears  to  reveal  the  degree  of  functional  derangement 
in  nephritis,  whether  of  the  acute  or  chronic  variety.  It  has 
also  been  of  value  in  diagnosing  impending  uremia  and  dis- 
tinguishing uremia  from  conditions  simulating  it. 

LABORATORY    PROCEDURES. 

Of  the  various  tests  which  have  been  employed  for  the  detec- 
tion of  protein  substances  in  urine,  the  greater  number  involve 

1.  von  Noorden:  Metabolism  and  Practical  Medicine,  Eng.  Ed., 
1907,  II,  p.  477;  also  Post-Graduate,  1913,  XXVIII,  p.  3. 


54  PATHOLOGICAL  CHEMISTRY 

precipitation  reactions.  In  some  of  these  the  protein  is  pre- 
cipitated because  of  its  insolubility  in  the  reagent  employed; 
in  others,  by  the  action  of  the  reagent  to  form  an  insoluble 
compound  with  the  protein,  as  in  the  case  of  the  alkaloidal 
reagents.  In  performing  all  these  tests  it  is  essential  that  the 
urine  should  be  perfectly  clear.  If  it  is  not  clear,  nitration 
through  filter  paper  will  usually  suffice  for  clarification,  but  in 
case  this  is  ineffective,  the  urine  may  be  shaken  with  powdered 
magnesia,  or  kaolin  and  again  filtered. 

1.  Heller's  Test. — When  properly  applied,  this  test  is  one  of 
the  most  satisfactory  general  tests  we  possess.  About  one  cc. 
of  pure  nitric  acid  is  placed  in  a  small  test  tube.  By  means  of  a 
pipette  having  a  small  rubber  bulb  at  one  end  and  a  ragged  non- 
tapering  edge  at  the  other,  an  equal  amount  of  urine  may  be 
allowed  to  flow  down  the  side  of  the  tube  without  inclining  or 
removing  from  the  rack.  By  this  procedure,  a  perfect  stratifi- 
cation may  be  obtained  with  great  rapidity.  In  the  presence  of 
albumin  a  white  zone  of  precipitated  albumin  will  be  observed 
at  the  point  of  juncture  of  the  two  liquids.  If  the  albumin  is 
present  in  very  small  amount,  the  white  zone  may  not  form 
until  the  tube  has  been  allowed  to  stand  several  minutes.  With 
concentrated  urines,  uric  acid  or  urea  may  occasionally  cause 
confusion  to  the  inexperienced,  due  to  the  precipitation  of  uric 
acid  and  urates,  or  the  formation  of  urea  nitrate.  Simply 
diluting  the  urine  will  remove  these  difficulties.  The  fine  ring 
which  appears  in  the  clear  urine  above  and  separated  from  the 
albuminous  ring  is  generally  ascribed  to  the  presence  of  urates 
though  certain  investigators  regard  it  of  a  protein  nature.  After 
the  administration  of  certain  drugs,  a  white  precipitate  of  resin 
acids  may  form  at  the  contact  of  the  two  fluids.  This  being 
the  case  the  ring  will  dissolve  in  alcohol,  whereas  the  albumin 
ring  will  not  dissolve.  Biliary  urine  reacts  with  nitric  acid  con- 
taining a  little  nitrous  acid  to  give  the  play  of  colors  referable 
to  the  action  of  nitric  acid  upon  bilirubin. 

2.  Robert's  Test. — This  test  is  carried  out  in  the  same  manner 
as  Heller's  test,  except  that  Robert's  reagent1  is  substituted 
for  the  nitric  acid  of  the  previous  test.  With  this  test  colored 
rings  do  not  form  and  the  test  is  slightly  more  sensitive  than 


1.   The    reagent  is  prepared  by  mixing  five    parts  of  saturated  mag- 
nesium sulphate  and  one  part  cone,  nitric  acid. 


TESTS  FOR  ALBUMIN  55 

Heller's,  but  is  subject  to  the  same  disadvantage  in  that  nucleo- 
protein  and  mucin  are  precipitated. 

3.  Heat  Test. — From  10  to  15  cc.  of  clear  urine  are  placed  in  a 
i test  tube,  and  the  reaction  of  the  urine  tested.  If  it  is  no i:  faintly 
acid,  it  is  rendered  so  with  a  few  drops  of  very  dilute  (2  per 
cent.)  acetic  acid.  The  upper  part  of  the  tube  is  brought  to 
the  boiling  point,  when  in  the  presence  of  albumin,  a  white 
cloud  will  be  observed,  in  comparison  with  the  control  portion 
of  the  tube  below.  The  cloud  may  in  part  be  due  to  nucleo- 
protein  and  mucin,  but  the  addition  of  one  sixth  volume  of 
saturated  sodium  chloride  and  5  drops  of  50  per  cent,  acetic  acid, 
with  the  subsequent  boiling  of  the  upper  part  of  the  tube  will 
serve  to  distinguish  it  from  these  two  proteins,  as  in  this  case  the 
nucleoprotein  and  mucin  are  not  precipitated.  The  test  carefully 
applied  in  this  way  is  very  reliable. 

4.  Sulpho salicylic  Acid  and  Trichloracetic  Acid  Tests. — These 
two  tests  may  be  mentioned  together  because  of  their  great 
delicacy  and  the  general  similarity  of  the  reactions.  The  sulpho- 
salicylic  acid  may  be  used  in  the  form  of  a  20  per  cent,  aqueous 
solution,  and  the  trichloracetic  as  a  saturated  aqueous  so- 
lution. The  clear  urine  is  stratified  upon  the  solutions  as  in 
Heller's  test.  Proteoses  (albumoses)  are  precipitated,  but 
dissolve  on  warming,  and  reappear  on  cooling.  In  concentrated 
urines,  urates  may  be  precipitated  with  the  trichloracetic  acid, 
but  this  can  be  avoided  by  the  dilution  of  the  urine.  In  the 
case  of  sulphosalicylic  acid,  uric  acid  and  the  resins  are  not 
precipitated.  If  a  delicate  test  is  desired  these  reagents  are 
particularly  valuable,  as  for  example  when  tube  casts  have 
been  observed,  though  previous  tests  for  albumin  have  been 
negative. 

5.  Quantitative  Estimation  of  Protein. — It  cannot  be  said  that 
we  possess  any  very  satisfactory  clinical  method  for  the  estimation 
of  protein  in  urine.  Accurate  results  may  be  obtained  by 
coagulating  the  protein,  filtering  through  a  weighed  Gooch 
crucible  with  asbestos  or  glass  wool  mat,  drying  and  again 
weighing,  but  this,  method  can  hardly  be  applied  in  ordinary 
routme  work.  The  method  of  Esbach  with  the  original  picric 
acid  solution  yields  fairly  satisfactory  results.  In  the  hands  of 
the  authors  Tsuchiya's  phosphotungstic  acid  reagent  has  not 
been  found  as  accurate  as  the  original  picric  acid  solution  when 


56  PATHOLOGICAL  CHEMISTRY 

employed  in  the  Esbach  albuminometer.  Still  another  method 
which  yields  fairly  reliable  results  under  properly  controlled 
conditions  is  the  method  of  Purdy,  in  which  the  albumin  is 
precipitated  by  acetic  acid  and  potassium  ferrocyanide  and 
the  precipitate  thrown  down  in  a  graduated  centrifuge  tube. 
It  is  very  difficult,  however,  to  keep  a  centrifuge  in  a  condition 
that  it  will  maintain  the  necessary  1500  revolutions  per  minute. 

a.  Esbach' s  Method. — Fill  the  albuminometer  to  the  mark 
"  U  "  with  urine  acidified  if  necessary  with  a  few  drops  of  dilute 
acetic  acid  and  add  Esbach's  reagent1  to  "  R."  The  specific 
gravity  of  the  urine  should  not  exceed  1 .008,  the  proper  density 
being  obtained  by  accurate  dilution  with  water.  Stopper  the 
tube,  invert  it  slowly  several  times  in  order  to  insure  thorough 
mixing  of  the  fluids,  and  set  aside  for  twenty-four  hours  at  a 
temperature  of  about  15°  C.  The  height  of  the  precipitate  on 
the  scale  indicates  directly  the  number  of  grams  of  dry  protein 
in  a  liter  of  the  urine.  In  case  it  is  desirable  to  ascertain  the 
quantity  of  albumin  at  once,  employ  Kwilecki's  modification  as 
follows:  add  10  drops  of  10  per  cent,  ferric  chloride  to  the  acid 
urine  before  introducing  the  Esbach  reagent,  warm  the  tube  and 
contents  in  a  water  bath  at  72°  C.  for  five  minutes  and  take  the 
reading. 

If  it  is  desired  to  employ  Tsuchiya's  phosphotungstic  acid 
reagent,2  this  may  be  used  in  the  same  tube  and  same  way  as  the 
Esbach  reagent. 

b.  Purely' s  Method. — Ten  cc.  of  clear  urine  are  placed  in  a 
15  cc.  graduated  centrifuge  tube,  3  cc.  of  10  per  cent,  potassium 
ferrocyanide  and  2  cc.  50  per  cent,  acetic  acid  added.  The  urine 
and  solutions  are  mixed,  the  tube  set  aside  for  10  minutes  to 
allow  precipitation  of  the  albumin  and  then  centrifugal] zed  for 
exactly  three  minutes  at  1500  revolutions  per  minute,  in  an  instru- 
ment with  a  radius,  including  the  tubes,  of  just  6|  inches.  The 
tube  is  then  removed  and  the  grams  protein  per  liter  read  off 
from  the  following  table  compiled  from  Purdy.3  When  the  amount 
of  protein  is  large  the  urine  should  be  accurately  diluted. 

1 .  The  reagent  is  composed  of  10  grams  of  picric  acid  and  20  grams  of 
citric  acid  dissolved  in  1000  cc.  of  distilled  water. 

2.  Tsuchiya's  reagent  consists  of  1.5  grams  of  phosphotungstic  acid 
dissolved  in  5  cc.  cone,  hydrochloric  acid  and  95  cc.  of  alcohol. 

3.  Purdy:  Practical  Urinalysis  and  Urinary  Diagnosis,  6th  edit., 
Philadelphia,   1901,  p.  80. 


TESTS  FOR  ALBUMIN 


57 


Volume  of  precipitate 
in  graduated  tube. 


Dry  weight  of  pro- 
tein to  liter 


Volume  of  precipitate    j  Dry  weight  of  pro- 
in  graduated  tube  tein  to  liter 


grams 


grams 


0.25 

0.5 

2.75 

5.7 

0.5 

1.0 

3.0 

6.3 

0.75 

1.6 

3  25 

6.8 

1.0 

2.1 

3.50 

7.3 

1.25 

2.6 

3.75 

7.8 

1.5 

3.1 

4.0 

8.3 

1.75 

3.6 

4.25 

8.9 

2.00 

4.2 

4.50 

9.4 

2.25 

4.7 

4.75 

9.9 

2.50 

5.2 

5.0 

10.4 

Detection  of  Other  Protein  Substances  in  Urine. — It  is  occasion- 
ally of  importance  to  examine  urine  for  other  protein  substances, 
aside  from  albumin  and  globulin.  Among  such  are  nucleo- 
protein,  hemoglobin,  "  Bence  Jones  "  protein,  proteoses  and 
amino  acids,  the  Bence  Jones  protein  being  very  rare,  and 
associated  with  -multiple  myeloma  of  the  bone. 

5.  Nucleo protein. — Nucleoprotein  cannot  be  positively  identi- 
fied in  urine,  especially  in  the  presence  of  other  protein  sub- 
stances without  considerable  difficulty.  If  urine  diluted  about 
three  times  shows  a  turbidity  when  made  strongly  acid  with 
acetic  acid,  it  indicates  the  presence  of  nucleoprotein.  If 
albumin  is  also  present  this  should  previously  be  removed  by 
boiling  and  filtering. 

Ott's  test  has  been  supposed  to  demonstrate  nucleoprotein. 
A  few  cc.  of  urine  are  mixed  with  an  equal  volume  of  saturated 
salt  solution  and  Almen's  reagent1  slowly  added.  A  bulky 
precipitate  appears  in  the  presence  of  nucleoprotein. 

6.  Detection  of  Proteoses — Method  of  Bang. — Ten  cc.  of  urine 
are  saturated  with  ammonium  sulphate  with  the  aid  of  heat 
(about  10  grams  required)  and  then  brought  to  boiling.  The 
precipitate  is  thrown  down  by  centrifuging,  then  rubbed  up  in 
a  mortar  with  96  per  cent,  alcohol  to  remove  urobilin.  The 
alcohol  is  poured  off  and  the  residue  treated  with  distilled  water, 
warmed  and  filtered.  The  filtrate  contains  the  proteose  which 
will  be  shown  by  the  biuret  reaction,  i.e.,  the  addition  of  strong 

1 .  Prepared  by  dissolving  five  grams  of  tannin  in  240  cc.  of  50  per  cent, 
alcohol  and.  add  10  cc.  of  25  per  cent,  acetic  acid. 


58  PATHOLOGICAL  CHEMISTRY 

caustic  alkali  followed  by  a  few  drops  of  very  dilute  copper 
sulphate,  resulting  in  the  production  of  a  pink  color  in  the 
presence  of  proteoses. 

7.  Amino  Acids. — Very  recently  Benedict  and  Murlin1  in  a 
preliminary  note  have  suggested  a  simple  modification  of  the 
Henriques-Sorensen  formol  titration  for  amino  acids  by  which 
they  may  be  titrated  directly.  The  ammonia  and  certain  of 
the  other  urinary  compounds  except  the  urea  are  removed  from 
the  urine  by  precipitation  with  10  per  cent,  phosphotungstic 
acid  in  a  strongly  acid  solution,  allowing  24  hours  for  sedimen- 
tation. The  excess  of  phosphotungstic  acid  is  now  removed  by 
means  of  tribasic  lead  acetate  and  litharge.  The  amino  acids 
are  now  titrated  in  the  filtrate  from  the  above,  after  the 
removal  of  the  excess  of  lead,  by  the  formalin  titration  method 
as  previously  described  for  ammonia.2 

8.  Estimation  of  Renal  Efficiency*  with  Phenols ulphonephtha- 
lein. — Rowntree  and  Geraghty4  have  recently  proposed  a 
simple  permeation  test  which  appears  to  be  very  efficient. 
The  test:  20  to  30  minutes  before  administering  the  drug,  the 
patient  is  given  200  to  400  cc.  of  water  to  insure  copious  urinary 
secretion.  Under  aseptic  precautions,  a  catheter  is  introduced 
into  the  bladder  and  the  latter  completely  emptied.  Noting  the 
time,  one  cc.  of  a  carefully  prepared  solution  of  phenolsulphone- 

1.  Benedict  and  Murlin:     Proc.  Soc.  Exp.  Biol.  Med.,  1912,  IX,  p.  109. 

2.  Consult  Chapter  III,  page  43. 

3.  Mention  should  also  be  made  of  the  very  interesting  and  promising 
work  of  Schlayer  and  his  pupils  in  Romberg's  Clinic  (see  papers  in  recent 
vols,  of  Deutsch.  Arch.  f.  klin.  Med.)  From  the  results  of  their  experi- 
mental and  clinical  work  these  investigators  are  of  the  opinion  that 
sodium  chloride  and  potassium  iodide  are  excreted  by  the  epithelium  of 
the  renal  tubules,  and  that  lactose  and  water  are  excreted  by  the  glomer- 
uli. They  believe  that  in  this  way  one  may  differentiate  renal  diseases 
with  predominantly  tubular  lesions  from  those  with  predominantly 
vascular  lesions.  For  discussion  see  Schlayer:  Medizinischen  Klinik, 
1912,  VIII;  Eppinger  and  Barrenscheen:  Wien.  klin.  Wochenschr.,  1912, 
LXII;  also  Baright:  Post-Graduate,  1913,  XXVIII,  p.  317,  who 
gives  simplified  technique  of  tests.  Rowntree,  Fitz  and  Geraghty 
{Arch.  Int.  Med.,  1813,  XI,  pp.  121  and  258)  in  recent  experiments  con- 
firm the  value  of  lactose  as  a  very  delicate  diagnostic  test,  though 
they  do  not  regard  the  sodium  chloride  and  potassium  iodide  tests  with 
especial  favor, 

4.  Rowntree  and  Geraghty:  Jour.  Pharmacol.  Exper.  Therap.,  1910, 
I,  p.  579;  Jour.  Amer.  Med.  Assoc,  1911,  Vol.  LVII,  p.  811;  Arch. 
Int.  Med.,  1912,  IX,  p.  284. 


TESTS  FOR  RENAL  EFFICIENCY  59 

phthalein,  containing  6  mgms.  to  the  cc.,  (may  be  obtained 
already  prepared  in  the  form  of  ampoules)  is  carefully  adminis- 
tered subcutaneously  or  preferably  intramuscularly  in  the 
lumbar  muscles  by  means  of  an  accurately  graduated  syringe. 
The  urine  is  now  allowed  to  drain  into  a  test-tube  in  which  has 
been  placed  a  drop  of  25  per  cent,  sodium  hydroxide  and  the 
time  of  the  appearance  of  the  first  pinkish  tinge  noted.  A 
rough  estimate  of  the  time  of  appearance  can  be  made  by  having 
the  patient  void  urine  at  frequent  intervals  without  the  use  of 
a  catheter.  Where  the  catheter  has  been  employed  and  there 
is  no  urinary  obstruction,  the  catheter  is  withdrawn  at  the  time 
of  the  appearance  of  the  drug  in  the  urine,  and  the  patient  is 
instructed  to  void  into  one  receptacle  at  the  end  of  one  hour 
and  into  another  receptacle  at  the  end  of  two  hours.  If  it  is 
desired  to  distinguish  between  the  efficiency  of  the  two  kidneys, 
the  urine  may  be  collected  separately  from  each  kidney  by 
catheterizing  the  ureters.  The  drug  normally  appears  in  the 
urine  in  five  to  ten  minutes,  38  to  60  per  cent,  being  eliminated 
the  first  hour  and  60  to  85  per  cent,  during  the  two  hours.  The 
test  is  of  particular  value  in  determining  the  degree  of  functional 
derangement  in  nephritis,  whether  of  the  acute  or  chronic 
variety,  also  in  uremia,  etc.  The  percentage  elimination  is 
estimated  by  treating  the  urine  of  the  two  periods  with  sufficiently 
strong  sodium  hydroxide  to  produce  the  maximum  red  color 
and  each  diluted  to  1000  cc,  if  the  depth  of  the  color  will  allow. 
This  is  then  compared  in  a  Duboscq  colorimeter  with  a  standard 
solution  containing  3  mgms.  of  the  drug  to  the  liter,  the  control 
prism  being  set  at  10  mm.  (Rowntree  and  Geraghty  now  employ 
the  cheaper  Hellige  instrument).  With  a  colorimeter,  the 
determination  of  the  percentage  elimination  is  very  simple  and 
rapid.  A  series  of  ten  test  tubes  containing  solutions  of  phenol- 
sulphonephthalein  of  different  known  concentration  may  be 
prepared,  the  equivalents  of  5,  10,  15,  20,  25,  30,  35,  40,  45  and 
50  per  cent,  of  the  injected  drug,  with  which  the  drug  excreted 
in  the  urine,  when  diluted  as  above,  may  be  measured.  These 
standard  solutions  will  keep  for  a  considerable  length  of  time  if 
an  excess  of  alkali  has  been  added  and  the  test  tubes  stoppered 
and  sealed  with  paraffin.  By  this  method  the  quantitative  ex- 
cretion of  the  drug  may  be  approximately  ascertained,  probably 
within  5  per  cent,  of  the  correct  value,  and  will  obviate  the 
need  for  a  colorimeter. 


CHAPTER  V. 
Glucosuria  and  Other  Types  of  Mellituria. 

The  elimination  of  appreciable  quantities  of  sugar  in  the  urine 
is  evidence  of  some  inefficiency  of  carbohydrate  metabolism  or 
indiscretion  of  diet.1  The  metabolic  defect  may  be  traced  to 
lesions  of  certain  organs,  e.g.,  the  pancreas,  or  to  abnormal  de- 
velopment of  other  glands  such  as  the  thyroids  or  hypophysis, 
while  not  infrequently  there  is  no  obvious  cause.  Sugars  other 
than  glucose  may  appear  in  the  urine.  Thus  in  certain  instances, 
arabinose,  levulose,  galactose,  lactose,  maltose  and  sucrose  may 
be  eliminated;  and  for  the  condition  characterized  by  the  pres- 
ence in  the  urine  of  a  sugar  without  specification,  the  term 
"  mellituria  "  is  appropriate. 

Since,  as  stated,  most  types  of  mellituria  are  results  of  devi- 
ations from  physiological  functions,  consideration  of  these 
conditions  will  be  preceded  by  a  brief  review  of  the  processes 
of  carbohydrate  metabolism  occurring  normally.  The  blood 
of  a  normal  individual  contains  from  .07  to  .11  per  cent,  of  glu- 
cose. Ordinarily  the  concentration  of  the  latter  does  not  exceed 
these  limits,  however,  great  or  small  the  carbohydrate  intake,  and 
is  essentially  independent  of  the  fuel  requirements  of  the  body. 
Should  the  mechanism,  by  which  this  constancy  is  maintained, 
become  defective,  glucose  will  accumulate  in  the  blood,  which 
condition  of  hyperglucemia  is  the  immediate  cause  of  glucosuria. 

Usually  the  body  is  supplied  with  glucose  at  intervals  during 
the  day,  the  amount  ingested  at  any  one  period  being  in  ex- 
cess of  the  immediate  fuel  requirements.  By  means  of  an  en- 
zymatic process  this  excess  is  converted  into  and  stored  as  gly- 
cogen in  the  liver  (glycogenesis),  the  reverse  action,  (glycogenol- 
ysis)  occurring  as  more  glucose  is  needed  for  combustion.  The 
storage  capacity  of  the  liver  for  glycogen  is  approximately  150 
to  200  grams;  and  should  this  limit  be  exceeded,  the  remaining 
sugar  would  be  retained  and  stored  as  glycogen  in  the  muscles. 


1 .  The  urine  of  a  normal  individual  may  contain  .01  to  .06  per  cent, 
of  glucose.  Such  small  quantities,  however,  cannot  be  detected  by  the 
usual  clinical  methods. 

60 


GLUCOSURIA  61 

The  concentration  oi  glycogen  in  the  muscle  tissue  may  reach  two 
per  cent,  and  in  special  cases  four  per  cent.,  corresponding  to 
sufficient  carbohydrate  to  maintain  body  heat  for  two  to  four 
days.  If  the  carbohydrate  intake  is  so  great  that  the  storage 
capacity  of  both  the  liver  and  muscles  is  overtaxed,  the  glucose 
not  converted  to  glycogen  is  further  prevented  from  giving  rise 
to  hyperglucemia  and  glucosuria  by  being  transformed  into 
fat.  A  large  amount  of  sugar  can  be  stored  in  this  manner. 
There  is,  likewise,  a  limit  to  fat  formation  and  should  this  point 
be  reached,  still  another  means  is  available  by  which  the  excess 
of  blood  sugar  may  be  removed  from  the  circulation — elimination 
through  the  kidneys.  When  all  the  storage  depots  become 
loaded  to  their  full  capacity,  the  sugar,  in  excess  of  that 
required  to  meet  the  fuel  needs  of  the  body  and  to  maintain 
essentially  constant  the  sugar  concentration  of  the  blood,  is 
excreted  in  the  urine.  Any  one  or  more  of  these  regulatory 
mechanisms  may  become  defective,  allowing  an  excess  of  sugar 
to  accumulate  in  the  blood  and  thus  give  rise  to  glucosuria.  Ex- 
amples of  such  defects  follow. 

Glucose  may  be  absorbed  from  the  alimentary  tract  and  be 
supplied  to  the  liver  more  rapidly  than  it  can  be  converted  into 
glycogen.  The  sudden  accumulation  of  sugar  in  the  blood 
would  lead  to  glucosuria — "  alimentary  glucosuria."  Norm- 
ally an  administration  upon  the  empty  stomach  of  100-200 
grams  of  glucose  will  be  tolerated,  and  this  serves  as  a  test  of 
the  power  of  glycogenesis.  Glucosuria  following  the  ingestion 
of  even  very  large  amounts  of  starch — '■  glucosuria  ex  arnylo  " — 
is  practically  never  observed  in  the  healthy  person.  There  are 
individuals  in  whom  the  function  of  glycogenesis  is  only  slightly 
impaired,  while  in  an  advanced  case  of  diabetes  it  is  almost 
completely  destroyed.  If  the  other  storage  places  are  efficient 
andif  the  power  of  burning  sugar  is  not  impaired,  there  may  be 
only  a  transient  glucosuria  as  a  result  of  the  defective  glyco- 
genesis. This  condition  is  presumably  to  be  attributed  to  the 
absence  or  insufficiency  of  an  active  glycogenetic  enzyme,  al- 
though it  may  be  due  primarily  to  disease  of  the  pancreas.  It 
is  interesting  to  note  that  even  in  advanced  cases  of  organic 
disease  of  the  liver  the  function  of  the  formation  of  glycogen 
from  glucose  is  retained,  although  frequently  in  these  conditions 
the  liver  is  unable  to  properly  utilize  levulose  and  hence,  ad- 


62  PATHOLOGICAL  CHEMISTRY 

ministration  of  this  sugar  often  gives  rise  to  levulosuria.  Glu- 
cosuria  is  noted  following  the  ingestion  of  moderate  doses  of  sugar 
in  gout,  obesity,  exophthalmic  goitre,  hypertrophic  cirrhosis 
of  the  liver,  fatty  liver,  pneumonia,  influenza,  alcoholism  and 
lead  poisoning. 

In  addition  to  defective  glycogenesis,  there  may  be  an  exag- 
geration of  the  function  of  glycogenolysis,  whereby  glycogen 
is  more  rapidly  transformed  into  glucose  than  it  is  needed  for 
combustion  and  also  in  excess  of  the  capacity  of  the  muscular 
system  to  reconvert  the  liberated  glucose  into  glycogen.  Con- 
sequently an  excess  of  sugar  finds  its  way  into  the  circulation 
and  glucosuria  results.  This  type  of  glucosuria  is  of  nervous 
origin  and  is  probably  due  to  stimulation  of  the  liver  cells  which 
elaborate  the  glycogenolytic  enzyme.  It  is  possible  that  the 
nervous  influences  bring  this  about  indirectly  by  primarily 
stimulating  the  adrenals  (see  p.  66.)  Experimentally,  gluco- 
suria may  be  evoked  in  the  rabbit  by  puncturing  the  floor  of  the 
fourth  ventricle  as  Claude  Bernard  showed  many  years  ago. 
Frequently  glucosuria  of  this  type  follows  traumatism  of  the 
nervous  system,  and  indirect  stimulation  of  any  part  of  the 
nervous  system,  if  sufficiently  strong,  may  produce  glucosuria. 
Fright  or  excitement  may  be  associated  with  the  appearance  of 
glucose  in  the  urine.1  Cases  of  nervous  glucosuria  are  not 
usually  persistent.  Under  this  head  would  appear  to  come 
the  glucosuria  resulting  from  the  administration  of  large  doses 
of  certain  poisons,  such  as  strychine,  morphine,  amyl  nitrite, 
prussic  acid,  ether,  chloroform,  carbon  monoxide,  and  many 
others.  Underhill2  has  called  attention  to  the  respiratory  dis- 
turbances accompanying  the  use  of  some  of  these  compounds, 
and  Henderson  and  Underhill  have  pointed  out  the  frequency 
with  which  acapnia  is  associated  with  glucosuria.3  They  be- 
lieve that  ether  glucosuria  is  due  to  acapnia,  which  also  is  usually 
the  cause  of  traumatic  and  emotional  glucosurias.  Henderson 
and  Underhill  further  observed  that  they  never  found  glucosuria 
in  dogs  which  had  been  brought  quietly  into  deep  ether  anes- 
thesia. 


1.  Cf.    Cannon,    Shohl    and    Wright:      Amer.    Jour.    Physiol.,    1911, 
XXIX,  p.  280. 

2.  Underhill:     Jour.  Biol.  Chem.,  1905,  I,  p.  124. 

3.  Henderson  and  Underhill:      Amer.  Jour.  Physiol.,   1911,    XXVIII, 
p.  275. 


GLUCOSURIA  63 

Glycogenes:s  may  be  reduced  and  glycogenolysis  may  be 
excessive  in  the  muscles  as  well  as  in  the  liver.  Either  of  these 
conditions  would  tend  to  produce  hyperglucemia  and  glucosuria. 
Both  of  these  abnormal  relations  between  sugar  and  glycogen 
in  the  muscles  are  present  in  diabetes. 

It  has  been  stated  that  a  large  amount  of  sugar  can  be  trans- 
formed into  and  stored  as  fat,  but  that  there  is  a  limit  to  this 
fat  formation.  It  is  apparent  that  very  obese  individuals  are 
living  close  to  this  limit,  since  a  slight  excess  of  carbohydrate 
in  the  diet  frequently  gives  rise  to  glucosuria.  On  the  other  hand 
there  are  individuals,  who  have  very  great  powers  of  combustion, 
and  they  store  fat  only  with  considerable  difficulty.  Advanced 
cases  of  diabetes  have  lost  not  only  the  power  of  burning  sugar  but 
also  the  ability  to  store  fat.  Occasionally  there  are  encount- 
ered diabetics  who  become  very  obese,  such  individuals  having 
apparently  retained  the  function  of  forming  fat  although  their 
sugar  burning  powers  are  reduced  to  a  minimum. 

In  addition  to  the  above  mentioned  types  of  glucosuria  there 
are  certain  others  less  well  denned,  such  as  the  glucosuria  of  ado- 
lescence and  the  glucosuria  associated  with  certain  skin  diseases. 

Hyperglucemia  is  the  immediate  cause  of  all  the  instances  of 
glucosuria  just  discussed.  In  a  condition  of  hyperglucemia  more 
sugar  is  present  in  the  blood  than  can  be  retained  by  the  kidneys, 
and  hence  glucosuria  results.  The  level  of  renal  retention  for 
sugar  may  be  raised  or  lowered.  In  certain  cases  of  diabetes, 
especially  when  complicated  with  nephritis,  this  level  may  be 
raised,  thus  permitting  a  marked  hyperglucemia  without  a 
correspondingly  strong  glucosuria.  On  the  other  hand,  there 
have  been  reported  cases  of  glucosuria,  which  could  most  readily 
be  explained  by  assuming  that  the  level  of  renal  retention  had 
been  lowered.1  Such  cases  of  "  renal  glucosuria  "  are  charac- 
terized by  no  hyperglucemia,  but,  nevertheless,  excrete  continu- 
ously over  long  periods  of  time  small  amounts  of  sugar;  and  the 
condition  is  apparently  uninfluenced  by  the  presence  or  absence 
of  carbohydrate  in  the  diet.  Experimentally  renal  glucosuria 
may  be  induced  by  injections  of  phlorhizin.  Following  the  use 
of  this  compound  there  are  observed  marked  hypoglucemia  and 
strong  glucosuria.     To  increased  permeability  of  the  kidney, 

1 .  For  descriptions  of  several  cases  of  glucosuria  of  this  type,  see  Gar- 
rod:    Lancet,  March  9,  1912,  p.  634. 


64  PATHOLOGICAL  CHEMISTRY 

Underhill  and  Closson  have  attributed  the  hypoglucemia  and 
glucosuria  resulting  from  injections  of  sodium  chloride  into  the 
venous  circulation  of  the  rabbit.1 

RELATION     OF     THE     INTERNAL     SECRETIONS     TO     CARBOHYDRATE 

METABOLISM.2 

Pancreas. — In  diabetes  the  underlying  disturbance  is  an  in- 
ability on  the  part  of  the  muscle  cells  to  burn  glucose,  and  is 
attributed  to  an  insufficiency  or  absence  of  the  internal  secre- 
tion of  the  pancreas.  It  is  believed  that  this  secretion  furnishes 
something  which  activates  the  glucolytic  enzyme  of  the  muscles, 
which  by  itself  is  unable  to  bring  about  the  combustion  of  sugar. 
Experiments  in  vitro  have  not  been  especially  confirmatory. 
When  a  mixture  of  muscle,  pancreas  and  glucose  is  allowed  to 
incubate,  there  is  indeed  a  diminution  in  the  glucose  content 
of  the  mixture,  but  the  presence  of  oxidation  products,  e.g., 
lactic  acid  and  alcohol,  cannot  be  detected.3  On  the  contrary, 
recent  studies  indicate  that  the  reaction  proceeds  in  the  other 
direction,  that  is,  glucose  is  transformed  into  maltose.3  The 
view  is  nevertheless  held  that  in  the  body,  the  internal  secretion 
of  the  pancreas  is  essential  to  carbohydrate  oxidation;  and  to 
the  islands  of  Langerhans  has  been  attributed  the  function  of 
elaborating  this  secretion.  From  the  intimate  relations  of  the 
pancreas  to  diabetes,  one  would  expect  to  find  distinct  lesions 
of  this  organ;  but  these  are  not  always  obvious.  Moreover, 
serious  but  more  or  less  localized  diseases  of  the  pancreas  are 
noted,  which  are  not  regularly  accompanied  by  glucosuria. 
Indeed,  when  it  is  recalled  that  a  large  part  of  the  pancreas 
may  be  removed  without  evoking  diabetes,  it  is  not  difficult  to 
understand  that  in  such  pancreatic  diseases  a  sufficient  number 
of  the  islands  of  Langerhans  may  have  remained  functionally 
active.  It  is  the  diffuse  type  of  pancreatic  disease  that  is  more 
constantly  associated  with  diabetes. 

Thyroids. — Exophthalmic  goitre  is  not  infrequently  associated 
with  diminished  tolerance  for  carbohydrates  or  in  fact  with  severe 
diabetes.     This  is  attributed  to  an  overactivity  of  the  thyroid 

1.  Underhill  and  Closson:    Amer.  Jour.  Physiol.,  1906,  XV,  p.  321. 

2.  For  an  interesting  description  and  discussion  of  cases,  see  Garrod: 
Lancet,  March  2  and  9,  1912,  o.  557  and  629. 

3.  Levene  and  Meyer:    Jour.  Biol.  Chetn.,  1912,  XL,  p.  356. 


GLUCOSURIA  65 

glands  whereby  an  excess  of  their  secretion  is  formed.  More- 
over, administration  of  thyroid  preparations  to  normal  indi- 
viduals likewise  leads  to  glucosuria.  The  suggestion  has  been 
made  that  the  excess  of  thyroid  secretion  inhibits  the  action 
of  the  pancreas  directly,  or  indirectly  by  stimulating  the  forma- 
tion of  adrenaline,  injection  of  which  is  known  to  produce  glu- 
cosuria. While  exophthalmic  goitre  (hyperthyroidism)  is 
accompanied  by  a  diminished  tolerance  for  carbohydrate,  myx- 
edema (hypothyroidism),  on  the  other  hand,  is  usually  associated 
with  increased  tolerance.  Individuals  with  myxedema  are  able 
to  utilize  quantities  of  carbohydrate,  which  would  be  sufficiently 
great  to  cause  alimentary  glucosuria  in  the  normal  person. 
This  increased  tolerance  for  carbohydrate  may  be  lowered  to 
the  normal  level  by  the  adminstration  of  thyroid  preparations. 
Although  usually  diminished  tolerance  for  sugar  is  a  concomitant 
of  hyperthyroidism  and  increased  tolerance  is  associated  with 
hypothyroidism,  there  are,  nevertheless,  cases  of  exophthalmic 
goitre,  in  which  glucosuria  cannot  be  demonstrated,  and  like- 
wise instances  where  myxedema  is  accompanied  by  diabetes. 
These  apparently  contradictory  findings  need  not  necessarily 
discredit  the  view  that  hyperthyroidism  and  hypothyroidism 
are  associated  respectively  with  diminished  and  increased  tol- 
erance for  carbohydrate.  A  case  of  myxedema  may  be  com- 
plicated with  a  diseased  pancreas,  and  the  ensuing  glucosuria 
may  persist  in  spite  of  the  diminished  activity  of  the  thyroid, 
which  would  lead  to  increased  tolerance,  were  the  pancreas  nor- 
mal. Furthermore,  it  is  not  impossible  that  the  inactivity  of 
the  thyroid  would  result  in  hypertrophy  of  the  pituitary  glands, 
and  the  secretion  of  the  latter,  as  will  be  noted  below,  lowers 
the  tolerance  for  carbohydrate.  The  condition  of  the  parathy- 
roids may  be  a  factor  of  importance,  since,  according  to  Eppinger, 
Falta  and  Rudinger,  their  removal  leads  to  diminished  toler- 


ance 


Hypophysis. — Just  as  exophthalmic  goitre  is  due  to  hyperac- 
tivity of  the  thyroid,  so  acromegaly  is  believed  to  be  the  result  of 
an  overdevelopment  of  the  pituitary  body.  Acromegaly,  like 
Graves'  disease,  is  accompanied  by  lowered  carbohydrate  tol- 
erance or  even  a  true  diabetes.     Out  of  176  cases  of  acromegaly, 

1.  Eppinger,  Falta  and  Rudinger:  Zeit.  f.  klin.  Med.,  1909,  LXVIJ,  p. 
380. 


66  PATHOLOGICAL  CHEMISTRY 

Borchardt  found  35  per  cent  to  be  glucosuric.1  It  occasionally 
happens  that  the  glucosuria  accompanying  hyperpituitarism 
ceases  for  a  considerable  length  of  time  or  even  permanently. 
This  has  been  accounted  for  by  assuming  that  the  disappearance 
of  glucosuria  is  coincident  with  the  change  from  a  condition  of 
hyperpituitarism  to  one  of  hypopituitarism,  which  is  character- 
ized by  increased  tolerance.  An  analagous  situation  is  en- 
countered in  the  case  of  the  thyroid,  where  exophthalmic  goitre 
is  followed  by  myxedema. 

Adrenals. — Injection  of  adrenaline  induces  hyperglucemia  and 
glucosuria,  but  an  undoubted  instance  of  glucosuria  due  to  dis- 
ease of  the  adrenals  is  rare.  It  has  been  suggested  that  an  ex- 
cessive formation  of  adrenaline  may  be  the  cause  of  certain 
mild  types  of  diabetes  of  later  life;  and  it  has  been  shown  by 
Cannon,  Shohl  and  Wright2  that  in  the  case  of  cats  which  have 
been  excited  by  a  barking  dog,  there  is  an  increased  concentra- 
tion of  adrenaline  in  the  blood,  and  this  is  accompanied  by  glu- 
cosuria. Porges3  has  demonstrated  the  condition  of  hypo- 
glucemia  following  adrenalectomy  and  in  Addison's  disease — 
presumably  a  case  of  deficient  adrenal  secretion.  Hypoglu- 
cemia  has  also  been  demonstrated  following  the  administration  of 
phosphorus4  and  hydrazine,5  both  of  which  drugs  have  been  as- 
sumed to  bring  about  this  result  by  inhibiting  the  formation  of 
adrenaline.  Underhill  and  Fine  have  noted  hypoglucemia  and  ab- 
sence of  glucosuria  in  dogs  under  the  influence  of  hydrazine 
even  after  extirpation  of  the  pancreas.6  This  is  in  accord  with 
the  observations  of  Eppinger,  Falta  and  Rudinger  that  in  cases 
of  Addison's  disease  the  carbohydrate  tolerance  is  abnormally 
high.7 

Glucosuria  of  Pregnancy. — There  are  instances  in  which 
pregnancy  is  associated  with  glucosuria,  the  latter  usually  dis- 
appearing after  parturition.  To  temporary  overactivity  of  the 
thyroid  or  pituitary  has  been  ascribed  this   type   of  glucosuria. 

1.  Borchardt:    Zeit.f.  klin.  Med.,  1908,  LXVI,  p.  332. 

2.  Loc.  cit. 

3.  Porges:    Zeit.f.  klin.  Med.,  1909,  LXIX,  p.  341. 

4.  Frank  and  Isaac:  Arch.  f.  exper  Path.  u.-Pharm.  1911,  LXIV,  p. 
274. 

5.  Underhill:    Jour.  Biol.  Chem.,  1911,  X,  p.  159. 

6.  Underhill  and  Fine:     Jour.  Biol.  Chem.,  1911,  X,  p.  271. 

7.  Eppinger,  Falta  and  Rudinger:  Zeit.f.  klin.,  Med.,  1908,  LXVI,  p. 
1;  ibid,  1909,  LXVII,  p.  380. 


GLUCOSURIA  67 

DIABETES    MELLITUS. 

Severe  diabetes  is  characterized  by  a  copious  excretion  of 
urine,  the  volume  being  roughly  proportional  to  the  amount  of 
sugar  eliminated.  An  output  of  three  to  six  liters  is  commonly 
observed  and  in  exceptional  cases  the  quantity  may  reach  ten 
liters  or  more,  the  highest  elimination  recorded  being  28  liters. 
In  diabetes,  the  volume  and  specific  gravity  do  not  present 
the  inverse  relationship  usually  observed  in  the  normal  urine. 
Thus  specific  gravities  of  1.025  to  1.046  may  be  noted  with 
volumes  of  two  to  ten  liters.  The  highest  specific  gravity 
recorded  is  1.074. 

A  more  detailed  consideration  should  be  accorded  the  subject 
of  diabetes  since  it  is  such  a  fundamental  disturbance  of  carbohy- 
drate metabolism  and  involves  such  important  changes  in  the 
metabolism  of  protein  and  fat.  Taylor  classifies  cases  of  diabetes 
under  three  heads,  essentially  as  follows:1 

a.  Cases  occurring  most  frequently  before  middle  life,  of 
relatively  rapid  onset,  and  usually  terminating  fatally.  They 
exhibit  all  the  typical  metabolic  derangements  of  diabetes. 
The  cause  is  difficult  to  ascertain.  Post  mortem,  lesions  of  the 
islands  of  Langerhans  are  usually  found. 

b.  Those  cases  characterized  at  first  merely  with  an  alimentary 
glucosuria,  but  which  gradually  pass  into  typical  diabetes,  and, 
excepting  their  more  chronic  nature,  differ  in  no  important  re- 
spect from  the  first  mentioned  type  of  diabetes.  Such  cases 
are  most  frequently  observed  after  middle  life.  Some  of  these 
cases  are  attended  with  all  the  metabolic  defects  of  diabetes, 
although  a  large  number  retain  at  least  the  function  of  fat  for- 
mation for  a  considerable  length  of  time. 

c.  Cases  of  diabetes  resulting  apparently  from  some  previous 
disease,  e.g.,  gout,  arteriosclerosis,  obesity,  or  cirrhosis  of  the 
liver.  A  small  percentage  of  the  cases  of  this  class  do  de- 
velop into  typical  diabetes,  but  this  is  not  true  of  the  majority. 
Glucosuria  may  persist  for  years  with  but  few  of  the  metabolic 
abnormalities  of  diabetes. 

It  is  among  cases  of  the  two  last  types  that  one  fails  to  obtain 
undoubted  evidence  of  pancreatic  lesions.      , 

The  principal  defect  of  metabolism  in  the  diabetic  is  the  in- 

1.  A.  E.  Taylor:  "  Digestion  and  Metabolism,"  Philadelphia  and  New 
York,  1912,  p.  291. 


68  PATHOLOGICAL  CHEMISTRY 

ability  to  burn  sugar.  As  already  described,  this  is  believed 
to  be  due  to  an  inefficiency  of  the  pancreas.  The  results  of 
extirpation  of  the  pancreas  closely  parallel  the  conditions  found 
in  human  diabetes.  Glycogenesis  is  reduced  and  glycogenolysis 
is  excessive  in  both  the  liver  and  muscular  tissue;  oxidation  of 
sugar  is  reduced  to  a  minimum ;  there  is  little  if  any  fat  formation, 
and  the  combustion  of  this  material  is  abnormal.  Thus  the 
factors  which  normally  cooperate  to  maintain  constant  the  sugar 
concentration  of  the  blood — however  great  may  be  the  ingestion 
of  carbohydrate— are  defective  in  diabetes.  The  sugar  which 
normally  should  be  stored  until  required  for  combustion  is  poured 
into  the  circulation,  giving  rise  to  hyperglucemia  and  glucosuria. 

As  previously  stated,  the  normal  concentration  of  sugar  in 
the  blood  varies  from  .07  to  .11  per  cent.  In  diabetes  sugar 
concentrations  of  .15  to  .25  per  cent,  are  not  uncommon  and  values 
as  high  as  1.0  per  cent,  have  been  recorded.  Since  hyperglucemia 
is  the  immediate  cause  of  glucosuria,  one  might  expect  these 
two  conditions  to  be  parallel  in  their  variations.  This,  however, 
is  not  always  the  case.  In  the  early  stages  of  diabetes,  a  slight 
hyperglucemia  may  be  associated  with  marked  glucosuria, 
an  indication  of  active  elimination  of  sugar  by  the  kidneys. 
In  the  later  stages  the  kidney  may  eliminate  sugar  less  readily, 
and  hence  a  strong  hyperglucemia  may  be  accompanied  by  but 
a  relatively  mild  glucosuria.  It  is  apparent  that  frequently  a 
blood  analysis  excells  an  examination  of  the.  urine  as  a  criterion 
upon  which  to  base  our  judgment  as  to  the  severity  of  the  dis- 
ease. It  may  be  noted  that  in  diabetes  complicated  with  neph- 
ritis one  frequently  observes  a  relatively  small  elimination  of 
sugar  in  the  urine. 

The  perfectly  healthy  active  individual  can  burn  as  much  as 
a  kilo  of  glucose  during  the  day.  Some  mild  cases  of  diabetes 
can  burn  100-150  grams  of  glucose  derived  from  ingested  car- 
bohydrate plus  the  sugar  formed  from  protein  of  the  diet  (about 
50  grams) .  It  may  be  pointed  out  that  certain  varieties  of  starch 
are  more  thoroughly  utilized  than  others.  Thus  the  diabetic 
tolerates  oat  meal  starch  very  much  more  readily  than  any 
other.  As  the  case  becomes  more  severe,  ingested  sugar  cannot 
be  burned  and  is  entirely  eliminated,  but  the  sugar  of 
protein  origin  is  still  available  for  combustion.  In  a  later 
stage  even  the  sugar  from  this  source  cannot  be  burned.     How- 


GLUCOSURIA  69 

ever,  if  the  protein  intake  be  low  and  the  fat  intake  high,  many 
of  these  cases  can  be  rendered  aglucosuric,  combustion  being  in 
great  part  supported  by  the  fat.  In  other  instances,  under 
no  circumstances  can  glucosuria  be  avoided.  Moreover,  as  the 
disease  becomes  more  severe,  less  reliance  can  be  placed  upon 
fat  combustion  as  this  is  defective.  Hence  greater  protein 
intakes  are  called  for. 

It  is  probably  true  that  in  the  last  stages  of  diabetes  there  is 
no  combustion  of  glucose  or  storage  of  glycogen  or  fat,  yet  all 
of  these  functions  may  be  retained  in  part  for  a  considerable 
period  of  time.  Occasionally  the  ability  to  form  fat  is  retained 
even  in  severe  diabetes,  resulting  in  obesity;  but  later  this  func- 
tion also  is  lost,  and  one  observes  the  characteristic  condition  of 
emaciation.  The  curve  of  development  of  a  case  of  diabetes 
does  not  always  follow  a  perfectly  regular  course.  There  are 
days  during  which  a  relatively  good  storage  of  glycogen  is  af- 
fected, only  to  be  followed  by  less  fortunate  days.  This  occasional 
recovery  of  glycogenesis  is  to  be  welcomed  since  it  spares  the 
burning  of  fat.  When  the  glycogen  reserves  are  reduced  to  a 
very  low  ebb,  combustion  of  fat  becomes  a  necessity,  and  since 
this  is  frequently  defective,  there  is  the  imminent  danger  of  a 
severe  or  even  fatal  acidosis.1 

PROTEIN    METABOLISM    IN    DIABETES. 

The  total  heat  production  of  the  diabetic  is  not  below  that 
found  in  a  normal  individual — in  fact  it  is  slightly  greater. 
This  is  of  interest  in  view  of  the  important  source  of  heat  lost 
in  the  sugar  eliminated  in  the  urine.  The  loss  is  made  good  by 
the  combustion  of  protein  and  fat ;  and  the  smaller  the  proportion 
of  utilizable  sugar  obtained  from  the  protein,  the  greater  must  be 
the  intake  of  the  latter  in  order  that  nitrogenous  equilibrium 
may  be  maintained.  On  account  of  loss  of  glucose  derived  from 
protein,  the  calorific  value  of  the  latter  may  be  reduced  50  per 
cent.  The  presence  of  a  suitable  quantity  of  protein  in  the  diet 
is  desired,  since  in  the  absence  of  sufficient  of  this  exogenous 
material,  the  body  will  draw  upon  its  own  tissue  protein  in  order 
to  maintain  proper  heat  production.  A  normal  individual  can 
maintain  nitrogen  equilibrium  on  as  little   as  7  grams  or  less  of 

1.  A  more  detailed  consideration  of  acidosis  will  be  presented  in  the 
following  chapter. 


70  PATHOLOGICAL  CHEMISTRY 

nitrogen  daily,  provided  that  sufficient  carbohydrate  and  fat  are 
also  ingested,  these  latter  materials  acting  as  protein  sparers. 
In  a  severe  case  of  diabetes,  however,  where  the  ability  to  burn 
sugar  is  greatly  reduced  and  likewise  fat  combustion  is  abnormal, 
a  greatly  increased  ingestion  of  nitrogen  will  be  required.  An 
intake  of  20  or  even  30  grams  of  nitrogen  may  be  needed,  and 
in  very  severe  cases  it  is  practically  impossible  to  maintain  nitro- 
genous equilibrium  however  great  the  nitrogen  intake  may  be. 

Reference  has  already  been  made  to  the  derivation  of  sugar 
from  protein.  A  brief  consideration  of  this  process  follows. 
As  described  in  an  earlier  chapter,  protein  during  digestion  is 
hydrolized  into  amino  acids.  After  absorption  the  latter  undergo 
a  further  cleavage  into  a  nitrogenous  fraction — ultimately 
eliminated  in  great  part  as  urea;  and  a  non-nitrogenous  fraction, 
which  is  in  part  burned  directly  and  in  part  converted  into  dex- 
trose. We  may  be  more  specific.  It  is  possible  to  state  which 
amino  acids  are  burned  directly,  and  which  are  converted  into 
dextrose.  It  has  been  shown  that  leucine,  tyrosine  and  pheny- 
lalanine are  oxidized  directly  through  the  diacetic  acid  stage 
(see  p.  79) ;  while  glycine,  alanine,  valine,  aspartic  acid,  gluta- 
minic  acid,  histidine,  proline  and  arganine  can  give  rise  to  dex- 
trose in  the  diabetic  organism.1  Normally  this  dextrose  is 
burned  or  stored  as  glycogen  or  fat,  but  in  severe  diabetes  it  is 
almost  completely  eliminated  in  the  urine.  Calculations  reveal 
the  fact  that,  were  all  the  carbon  of  the  protein  converted  into 
glucose,  one  gram  of  protein  nitrogen  would  be  equivalent  to 
eight  grams  of  glucose,  the  M  glucose:  nitrogen  ratio  "  being  8:1. 
As  a  matter  of  fact  experimental  studies  indicate  that  not  more 
than  4.5  grams  of  glucose  are  derived  from  the  protein  equivalent 
of  one  gram  of  nitrogen,  yielding  a  G:N  ratio  of  4.5:1.  The 
remainder  of  the  carbonaceous  derivatives  of  the  protein  is 
burned  directly.  In  human  diabetes  Lusk  has  frequently  found 
a  G:N  ratio  of  3.65:1,  which  he  has  regarded  as  the  "  fatal 
ratio;"  i.e.,  when  the  case  has  progressed  so  far  that  as  much  as 
3.65  grams  of  glucose,  formed  from  the  equivalent  of  one  gram 
of  nitrogen ,  are  excreted ,  the  prognosis  is  considered  very  bad .  The 
estimation  of  this  ratio  for  a  diabetic  on  a  protein-fat  diet  is 
obviously  of  great  importance,  since  the  finding  of  a  smaller 

1.    Dakin:  "  Oxidations  and  Reductions  in  the  Animal  Body,"  1912 
p.  58;  also  Jour.  Biol.  Chem.,  1913,  XHI,  p.  513. 


GLUCOSURIA  71 

ratio  is  evidence  that  some  of  the  sugar  of  protein  origin  is  being 
burned.  The  progressive  lowering  of  this  ratio  is  to  be  taken  as 
a  favorable  prognostic  sign.  Clinically,  ratios  as  high  as  8:1,  12:1 
or  even  14:1  have  been  reported.  Since  these  ratios  account  for 
more  glucose  than  could  possibly  have  been  derived  from  protein, 
the  assumption  has  been  made  that  fat  was  the  source  of  this 
extra  sugar.  As  a  matter  of  fact  these  results  cannot  be  accepted 
as  evidence  of  the  conversion  of  fat  into  sugar  since  adequate 
controls  were  frequently  lacking.  In  order  that  the  estimation 
of  this  ratio  may  be  of  value,  it  is  necessary  that  the  observation 
be  carried  over  several  days,  the  nitrogen  intake  accurately 
known,  and  that  the  diet  consist  of  protein  and  fat  only.  Proteins 
of  various  origins  differ  in  their  ability  to  promote  glucosuria. 
Thus  meat  is  most  potent  in  this  respect,  next  in  order  being 
casein,  egg  albumin  and  vegetable  proteins.1  It  is  possible  that 
the  nature  of  amino  acids  of  the  different  proteins  may  be  the 
determining  factor. 

As  above  indicated,  the  ability  to  burn  sugar  is  not  always 
entirely  lacking,  and  the  estimation  of  the  G:N  ratio  was  sug- 
gested as  a  method  for  determining  the  extent  to  which  this 
function  was  retained.  Another  means  of  learning  this,  and  also 
of  throwing  light  upon  the  character  of  metabolism  in  general, 
is    the    estimation    of    the    "  respiratory    coefficient,"    that    is 

expired  carbon  dioxide      _  ■  . 

: ; — -z — .     bor    the    combustion    of    sugar,  this 

inspired  oxygen  b 

quotient  is  1;  for  the  combustion  of  fat,  it  is  approximately  0.7 
and  in  the  case  of  protein  the  quotient  is  about  0.8.  In  dia- 
betes, quotients  have  been  obtained  varying  from  0.64  to  0.76. 
In  general  it  may  be  said  that  as  the  case  becomes  more  severe 
the  quotient  will  become  progressively  lower  although  to  this 
there  are  notable  exceptions.  For  a  more  comprehensive  dis- 
cussion of  this  phrase  of  the  subject,  reference  must  be  made  to 
other  works.2 

For  the  mild  or  moderately  severe  case  of  diabetes,  dietetic 
treatment  offers  some  hope  of  at  least  a  temporary  improvement, 
but  for  the  very  severe  case,  the  situation  is  indeed  a  delicate 

1 .  von  Noorden:  New  Aspects  of  Diabetes,  '*  Post-Graduate  Lectures, " 
New  York,  1912,  p.  18. 

2.  Cf.  A.  E.  Taylor:  loc.  cit.,- p.  335;  also  Benedict  and  Joslin:  Car- 
negie Inst.  Washington,  1910,  Pub.  No.  136. 


72  PATHOLOGICAL  CHEMISTRY 

one.  The  abnormally  high  concentration  of  sugar  in  the  cir- 
culation is  detrimental  to  the  tissues.  Resistance  is  lowered 
and  the  patient  not  infrequently  succumbs  to  tuberculosis  or 
pneumonia.  Wounds  heal  with  difficulty  and  infections  often 
terminate  in  gangrene.  Neuritis,  cataract  of  the  eye,  stomatitis, 
caries  of  the  teeth,  and  furunculosis  are  also  frequent  afflictions  of 
the  diabetic.  If  the  attempt  is  made  to  reduce  the  hypergluce- 
mia  by  lowering  the  protein  intake,  there  is  the  danger  that  the 
body  will  draw  upon  its  own  tissue  protein  to  meet  the  fuel  re- 
quirements, thus  aggravating  the  condition  of  emaciation.  More- 
over, in  such  a  case  a  greater  fat  ingestion  would  be  called  for 
and  since  the  combustion  of  this  material  is  incomplete,  there 
is  the  possibility  of  the  development  of  acidosis  with  the  danger 
of  an  impending  fatal  coma.  •  However,  in  spite  of  this,  it  has 
been  found  beneficial  occasionally  to  introduce  a  day  of  starva- 
tion, as  the  resulting  diminished  hyperglucemia  and  glucosuria 
induce  a  temporary  improvement  in  the  tolerance  for  carbohy- 
drate. 

OTHER    TYPES    OF    MELLITURIA. 

Pentosuria. — Pentosuria,  a  rare  condition,  the  cause  of  which 
is  obscure,  it  is  an  anomaly  of  metabolism  which  tends  to  run 
in  families,  but  is  probably  harmless.  The  pentose  found  in 
the  urine  is  optically  inactive  arabinose.  A  small  amount  of 
pentose  has  been  detected  in  the  urine  of  some  typical  cases  of 
diabetes.  Pentosuria  is  to  a  large  extent  independent  of  the 
presence  or  absence  of  pentoses  in  the  diet.  This  is  true,  at  least, 
of  the  types  of  pentosuria  just  described  which  are  probably  idio- 
pathic. However,  certain  cases  of  pentosuria  have  been  reported 
which  most  likely  owe  their  origin  to  pentoses  of  the  diet.1 
Cherries  and  apples  and  other  fruits  have  been  known  to  give 
rise  to  pentosuria  where  the  urine  had  been  previously  free 
from  pentose. 

Levulosuria. — Levulose — ingested  in  fruits  or  resulting  from 
cleavage  of  cane  sugar — is  converted  into  glucose  in  the  intestinal 
wall  and  liver.  The  ability  of  the  liver  to  affect  this  transforma- 
tion may  be  reduced,  in  which  condition  levulose  may  appear 
in  the  urine.  In  diabetes,  the  ingestion  of  levulose  will  exag- 
gerate the  glucosuria,  but  levulose  itself  may  not  be  excreted 

1.  Cf.  Neubauer-Huppert:  "  Analyse  des  Hams"  Wiesenbaden,  1910, 
p.  356. 


MELLITURIAS  73 

in  even  severe  cases  of  diabetes,  as  apparently  the  liver  retains 
for  a  long  time  the  function  of  converting  levulose  to  glucose. 
Evidence  of  levulosuria  is  therefore  to  be  regarded  as  an  unfavor- 
able diagnostic  sign,  since  it  points  to  an  additional  hepatic  de- 
fect. Levulosuria  has  been  observed  in  a  large  number  of  in- 
stances of  cirrhosis  of  the  liver,  although  it  is  not  confined  to 
injury  of  this  organ.  The  appearance  of  levulose  in  the  urine 
following  the  ingestion  of  a  definite  quantity  of  this  sugar 
(usually  100  grams)  is  regarded  as  a  valuable  test  for  chronic 
degenerative  processes  in  the  liver  such  as  cirrhosis.  Levulu- 
suria  has  also  been  reported  in  cases  of  pregnancy  and  after  ad- 
ministration of  thyroid  preparations ;  and  a  few  instances  of  ap- 
parently idiopathic  levulosuria  have  been  recorded. 

Galactosuria. — As  far  as  we  are  aware,  idiopathic  galactosuria 
does  not  occur,  but  an  alimentary  galactosuria  may  be  observed 
following  the  ingestion  of  a  sufficiently  large  amount  of  galactose 
or  lactose,  from  which  galactose  is  formed  during  digestion. 
Even  in  healthy  individuals,  the  administration  of  40  to  100 
grams  of  galactose  may  be  followed  by  galactosuria.  In  cirrho- 
sis of  the  liver  the  tolerance  is  said  to  be  lowered,  as  little  as  20 
grams  of  galactose  leading  to  galactosuria.  It  has  been  stated 
by  some  workers  that  the  sugar  frequently  appearing  in  the  urine 
of  children  suffering  with  intestinal  disturbances  is  galactose 
together  with  lactose.1  In  diabetes,  galactose  intensifies  the 
elimination  of  glucose  in  the  urine. 

Lactosuria. — Excepting  glucosuria,  lactosuria  is  perhaps  the 
most  common  type  of  mellituria.  Like  cane  sugar,  lactose  itself 
is  not  utilizable  by  the  body.  It  must  be  subjected  to  digestive 
action  whereby  it  is  converted  into  glucose  and  galactose.  Ali- 
mentary lactosuria  may  be  easily  produced  by  the  ingestion  of 
a  large  quantity  of  lactose  on  an  empty  stomach,  although  nor- 
mally such  a  condition  is  not  observed  after  partaking  of  even 
large  volumes  of  milk.  Gastro-intestinal  disturbances  in  child- 
ren are  not  infrequently  accompanied  by  lactosuria.  Lactose 
is  formed  in  the  mammary  glands.  If  its  elimination  is  impeded, 
it  is  forced  back  into  the  circulation  and  excreted  in  the  urine, 
since  lactose  is  not  available  to  the  organism.  Lactosuria  may 
ensue  during  the  last  months  of  pregnancy,  but  more  frequently 
during   the   early   period   of   lactation.      The  excretion  of  this 

1.    Cf.  Neubauer-Huppert:  loc.  cit.,  p..  144. 


74  PATHOLOGICAL  CHEMISTRY 

sugar  usually  ceases  after  lactation  has  been  established,  but  may 
appear  for  a  time  when  nursing  is  suspended. 

Saccharosuria. — It  is  only  after  conversion  into  glucose  and 
levulose  during  digestion  that  cane  sugar  becomes  available  to 
the  organism.  Should  cane  sugar  be  ingested  in  such  quantities 
that  it  is  absorbed  before  sufficient  time  has  elapsed  for  this 
transformation,  it  would  appear  in  the  urine.  Usually  as  much 
as  200  or  300  grams  of  cane  sugar  may  be  taken  without  pro- 
voking more  than  a  trace  of  saccharosuria. 

Maltosuria. — Maltosuria,  sometimes  associated  with  gluco- 
suria,  occurs  following  the  ingestion  of  excessive  amounts  of  mal- 
tose. This  sugar,  unlike  sucrose  or  lactose,  can  be  converted 
to  glucose  by  most  of  the  tissues.  Maltosuria  has  been  observed 
in  a  few  cases  of  diabetes. 

LABORATORY    PROCEDURES. 

1.  Reduction  Tests. — The  property  possessed  by  glucose,  in 
common  with  many  other  sugars,  of  taking  up  oxygen  in  alkaline 
solution,  especially  in  alkaline  copper  solutions,  has  been  generally 
employed  as  a  means  of  detecting  this  substance  in  urine.  Fehl- 
ing's  has  been  the  most  commonly  employed  solution  since 
its  introduction  more  than  sixty  years  ago.  This  solution  has 
recently  been  modified  by  Benedict1  so  as  to  greatly  increase 
its  delicacy  and  render  permanent .  its  keeping  power.  The 
strong  alkali,  potassium  hydroxide,  has  been  replaced  by  sodium 
carbonate  which  does  not  exert  upon  glucose  the  destructive 
action  of  the  hydroxide.  By  replacing  the  Rochelle  salt  with 
sodium  citrate  the  solution  has  been  found  to  keep  perma- 
nently. It  is  about  ten  times  as  sensitive  to  sugar  in  urine  as 
Fehling's  or  Haines'  solutions  but  unlike  these  latter  solutions, 
not  appreciably  reduced  by  creatinine,  uric  acid,  chloroform,  or 
the  simple  aldehydes.  When  albumen  is  present  in  the  urine, 
it  is  advisable  to  remove  it  by  coagulation  and  filtration  before 
applying  either  Fehling's  or  Benedict's  test.  The  removal  of 
albumen  is  essential  in  the  case  of  Nylander's  test,  as  this  reagent 
gives  a  change  of  color  with  albumen  similar  to  that  with  sugar. 

1.  Benedict:  Jour.  Biol.  Chem.,  1909,  V.  p.  485;  Jour.  Amer.  Med. 
Assoc,  1911,  LVII,  p.  1193;  Myers:  Munch,  med.  Wochenschr.,  1912, 
LIX,  p.  1494. 


TESTS  FOR  GLUCOSE  75 

a.  Benedict's  Test. — About  5  cc.  of  the  reagent1  are  placed 
in  a  test  tube  and  8  to  10  drops  (not  more)  of  the  urine  to  be  ex- 
amined added,  and  the  mixture  boiled  vigorously  from  one  to 
two  minutes.  It  is  allowed  to  cool  spontaneously.  In  the  pres- 
ence of  dextrose,  the  entire  body  of  the  solution  will  be  filled  with 
a  precipitate,  which  may  be  red,  yellow  or  green  in  color, 
depending  upon  the  amount  of  sugar  present.  If  the  amount 
of  glucose  be  small  (under  0.3  per  cent.)  the  precipitate  forms 
only  on  cooling.  If  no  sugar  be  present,  the  solution  either 
remains  perfectly  clear  or  shows  a  faint  turbidity  that  is  blue 
in  color  and  consists  of  precipitated  urates,  which  need,  however, 
cause  no  confusion.  Even  very  small  quantities  of  dextrose 
in  urine  (0.1  per  cent.)  yield  precipitates  of  surprising  bulk  with 
this  reagent. 

b.  Fehling's  Test. — Equal  parts  of  the  two  solutions2  are 
mixed  together  and  preferably  diluted  with  2  to  3  parts  of 
water.  About  5  to  10  cc.  of  this  solution  are  placed  in  a  test  tube, 
heated  to  boiling  and  8  to  10  drops  of  urine  added.  If  no  reduction 
occurs,  boil  again.  Allowances  must,  however,  be  made  with 
Fehling's  solution  for  reductions  from  uric  acid,  creatinine,  etc., 
which  are  especially  liable  to  occur  with  concentrated  urines 
when  the  solution  is  boiled,  subsequent  to  the  addition  of  the 
urine. 

c.  Nylander's  Test. — To  5  cc.  of  urine,  add  10  drops  of  Ny- 
lander's  reagent3  and  boil.  In  the  presence  of  sugar,  the  solu- 
tion turns  yellow,  and  finally  black,  bismuth  being  precipitated. 


1.  Benedict's  single  qualitative  solution  is  composed  of  17.3  grams 
of  copper  sulphate,  173.0  grams  of  sodium  citrate  and  100  grams  of  anhy- 
drous sodium  carbonate  (double  the  weight  of  the  crystalline  salt  may  be 
employed)  made  up  to  one  liter  with  distilled  water.  In  the  preparation 
of  the  solution,  the  copper  sulphate  should  be  dissolved  separately  in  about 
100-150  cc.  of  distilled  water  and  then  added  slowly  with  constant  stirring 
to  a  filtered  solution  (about  800  cc.)  of  the  other  ingredients  and  finally 
made  up  to  one  liter. 

2 .  Fehling's  solution  is  composed  of  two  solutions,  equal  parts  of  which 
are  mixed  as  used.  The  cupric  sulphate  solution  (a)  contains  34.65  grams 
of  cupric  sulphate  dissolved  in  water  and  made  up  to  500  cc,  and  the  alka- 
line tartrate  solution  (b)  125  grams  of  potassium  hydroxide  and  173  grams 
of  Rochelle  salt  dissolved  in  water  and  made  up  to  500  cc. 

3.  Nylander's  reagent  is  prepared  by  digesting  2  grams  of  bismuth 
subnitrate  and  4  grams  of  Rochelle  salt  in  100  cc.  of  a  10  per  cent,  solution 
of  potassium  hydroxide.    The  solution  is  then  cooled  and  filtered. 


76  PATHOLOGICAL  CHEMISTRY 

d.  Barfoed's  Test. — This  test  serves  to  detect  mono saccar ides. 
Place  about  5  cc.  of  Barfoed's1  solution  in  a  test  tube 
and  heat  to  boiling.  Add  the  urine  under  examination  slowly, 
a  few  drops  at  a  time,  heating  after  each  addition.  Reduction 
is  indicated  by  the  production  of  a  red  precipitate,  which  may 
not  form  until  the  tube  has  stood  a  few  minutes. 

2.  Phenylhydrazine  Reaction. — In  a  test  tube  prepare  a  mix- 
ture of  5  drops  of  phenylhydrazine  (the  base),  10  drops  of  glacial 
acetic  acid,  and  1  cc.  (20  drops)  of  a  saturated  solution  of  sodium 
chloride,  then  add  5  cc.  of  urine  and  boil  gently  for  a  few  minutes. 
In  the  presence  of  glucose,  yellow  phenylglucosazone  crystals 
will  appear  on  cooling,  which  may  be  readily  identified  under  the 
microscope  and  by  their  melting  point,  (slightly  above  200° 
C).  This  test  is  a  very  delicate  one  and  these  characteristic 
crystals  are  specific  of  glucose  (or  levulose),  hence  this  test 
is  a  very  important  one  when  it  is  desired  to  definitely  identify 
the  sugar. 

3.  The  Quantitative  Estimation  of  Glucose  in  Urine. — Three 
general  procedures  are  commonly  employed  clinically  for  the 
estimation  of  glucose  in  urine,  viz.,  titration,  fermentation,  and 
the  polariscopic  method.  The  titration  method  of  Benedict2, 
which  is  conceded  to  be  far  superior  to  the  older  titration  methods 
of  Fehling  and  Purdy,  is  perhaps  the  method  of  choice.  This 
method  as  we  have  found3  gives  very  excellent  results  and  no 
special  or  expensive  instrument  is  required.  It  is  superior  to 
the  Lohnstein  fermentation,  because  the  results  may  be  ob- 
tained at  once  (about  5  min.  necessary).  It  is  also  superior 
to  the  polariscopic  method  in  those  instances  when  levorotatory 
substances  (as  /3-hydroxybutyric  acid)  are  present,  thus  neces- 
sitating a  determination  both  before  and  after  fermentation. 

a.  Benedict's  Volumetric  Method. — The  procedure  which  has 
been  found  most  convenient  for  the  estimation  of  glucose  in 
urine  is  as  follows:  The  urine,  preferably  accurately  diluted, 
if  a  large  amount  of  glucose  is  indicated,  is  placed  in  an  accurately 
graduated  25  cc.   burette.     Twenty-five  cc.  of    the    volumetric 

1 .  Barfoed's  solution  is  composed  of  4.5  grams  of  neutral  crystallized 
cupric  acetate  dissolved  in  100  cc.  of  water  to  which  1.2  cc.  of  5  per  cent, 
acetic  acid  is  added. 

2.  Benedict:  Jour.  Biol.  Chem.,  1911,  IX,  p.  57;  Jour.  Amer.  Med. 
Assoc,  1911,  LVII,  p.  1193. 

3.  Myers:     Munch,  med.  Wochenschr.,  1912,  LIX,  p.  1494. 


ESTIMATION  OF  GLUCOSE  77 

solution1  are  pipetted  into  a  150  cc.  Jena  extraction  flask  as 
used  in  fat  extractions,  5-10  grams  of  sodium  carbonate  and  a 
bit  of  powdered  pumice  added.  The  mixture  is  heated  to  vig- 
orous boiling  on  a  wire  gauze  with  small  asbestos  mat  and  the 
urine  run  in  rapidly  until  a  chalk  white  precipitate  begins  to 
form  and  then  more  slowly  with  continuous  boiling,  until  one 
drop  dissipates  the  last  trace  of  blue  color,  indicating  the  end 
point.  Chloroform  must  not  be  present  in  the  urine  and  if  pres- 
ent, must  be  removed  by  boiling.  Benedict  found  that  25  cc. 
of  the  copper  solution  were  reduced  by  exactly  50  mgms.  of  glu- 
cose or  52  mgms.  of  levulose,  and  we  have  ascertained  that  the 
value  for  galactose  is  54  mgms.  and  for  lactose  67  mgms. 

b.  Polariscope  Examination. — To  estimate  the  amount  of 
glucose  in  urine  polariscopically  the  urine  must  be  perfectly  clear. 
Usually  this  is  not  the  case  and  to  clear,  shake  the  slightly  acid 
urine  with  a  pinch  of  basic  lead  acetate  and  then  filter.  Place 
the  clear  urine  in  the  polariscope  tube,  make  several  readings  and 
calculate  percentage  of  glucose  according  to  the  following  formula 

Observed  Rotation  X  100 
52.5*  X  length  of  tube      =  Percenta^e  of  ^osz 

c.  Fermentation  Method. — The  best  fermentation  instrument 
is  the  improved  instrument  of  Lohnstein.  This  instrument 
reads  to  10  per  cent,  of  sugar  and  has  two  scales,  one  for  room 
temperature  and  the  other  for  the  incubator.  One-half  cc.  of 
urine  is  carefully  placed  upon  the  clean  mercury  and  then  the 
same  amount  of  a  yeast  emulsion  is  added.  The  carefully  waxed 
stopper  is  then  inserted,  the  mercury  column  is  set  at  0,  the 
weight  placed  on  the  stopper  and  the  instrument  allowed  to 
incubate.  Very  accurate  results  are  obtained  with  this  instru- 
ment. The  only  disadvantages  are  the  length  of  time  required 
to  obtain  the  result  and  the  difficulty  in  cleaning  the  instrument. 

1 .  Benedict's  volumetric  solution  is  likewise  permanent  and  is  com- 
posed of  18.0  grams  of  copper  sulphate,  100  grams  of  anhydrous  or  double 
the  quantity  of  crystallized  sodium  carbonate,  200  grams  of  sodium  or 
potassium  citrate,  125  grams  of  potassium  sulphocyanate,  and  5  cc.  of 
5  per  cent,  potassium  ferrocyanide  solution,  made  up  to  one  liter  with  dis- 
tilled water.  In  preparation  the  ingredients  are  dissolved  in  the  same 
manner  as  the  qualitative  reagent,  i.e.,  the  copper  separately. 

2.  The  specific  rotation  of  d-glucose  may  be  taken  as  +52. 5°,  of  lac- 
tose as  -f53°,  of  d-galactose  as  +81°  and  of  levulose  as  — 92°,  Cf. 
Tollens:  Abderhalden's  Handbuch  der  Biochemichen  Arbeitsmethoden, 
1910,  II,  p.  122. 


78  PATHOLOGICAL  CHEMISTRY 

Occasionally  certain  other  substances  occur  in  urine  which 
may  cause  a  confusion  with  glucose  when  the  ordinary  copper 
solutions  are  the  only  source  of  data.  Among  such  are  levulose, 
lactose,  pentoses,  galactose,  conjugate  glucuronates,  homo- 
gentisic  acid,  etc.  Fermentation,  the  polariscope  and  the  phenyl- 
hydrazine  reaction  yield  especially  valuable  data  here.  Glucose 
and  levulose  are  both  fermentable  and  both  give  the  same  oza- 
zone.  Levulose,  however,  is  levorotatory  and  has  specific  re- 
actions— Borchardt's,  Seliawanoff's1.  Lactose  and  pentose 
are  not  directly  fermentable  with  yeast.  Lactose  does  not  give 
the  Barfoed  reaction,  being  a  disaccharide.  It  may  be  partially 
identified  by  the  mucic  acid  test2,  though  galactose  also  gives 
this  reaction.  Its  ozazone  is  not  sufficiently  insoluble  to  serve 
as  a  test  in  urine.  The  glucuronates  and  homogentisic  acid 
are  not  fermentable  with  yeast.  In  the  presence  of  glucuronates 
a  levorotation  will  be  observed  after  fermentation.  Pentoses 
and  the  glucuronates  both  give  Tollen's  phloroglucin  reaction,3 
but  the  pentoses  only  the  orcin  reaction,4  which  may  be  ap- 
plied in  the  form  of  Bial's  reagent.  A  urine  containing  homogen- 
tisic acid  does  not  give  the  bismuth  reaction,  and  furthermore , 
turns  dark  on  standing. 

1.  When  a  few  drops  of  urine  are  added  to  5  cc.  of  Seliawanoff's  re- 
agent, (0.05  grams  of  resorcin  in  100  cc.  of  dilute  (1-2)  hydrochloric  acid) 
and  the  mixture  boiled  a  red  color  and  a  red  precipitate  will  be  observed  in 
the  presence  of  levulose. 

2.  Upon  evaporating  100  cc.  of  urine  containing  lactose  with  20-25  cc. 
of  concentrated  nitric  acid  in  a  shallow,  broad  glass  vessel  over  a  water 
bath,  until  only  about  20  cc.  remain,  fine  gritty  crystals  of  mucic  acid 
should  separate  in  the  presence  of  lactose. 

3.  The  phloroglucin  reactio'n  is  performed  by  adding  a  little  of  the 
material  to  the  urine  mixed  with  an  equal  volume  of  hydrochloric  acid  (sp. 
gr.  1.09)  and  heating  on  the  water  bath.  Pentose,  galactose  or  glucuronic 
acid  will  be  indicated  by  the  appearance  of  a  red  color. 

4.  The  orcin  test  may  be  performed  in  a  similar  manner  to  the  phlor- 
glucin  reaction  substituting  orcin  and  heating  the  mixture  to  boiling  in 
this  case.  Here  the  end  color  reaction  is  green.  Bial  uses  as  reagent  30 
per  cent,  hydrochloric  acid  which  contains  one  gram  of  orcin  and  25  drops 
of  a  ferric  chloride  solution  (62.9  per  cent,  of  the  crystalline  salt)  in  500  cc. 
of  the  acid.  About  5  cc.  of  the  reagent  is  heated  to  boiling  and  then  a  few 
drops  (not  more  than  1  cc.)  of  the  urine  is  added  to  the  hot  but  not  boiling 
liquid.     In  the  presence  of  pentose  the  liquid  turns  a  beautiful  green. 


CHAPTER  VI. 

ACIDOSIS. 

In  the  course  of  the  metabolism  of  the  three  foodstuffs,  protein, 
carbohydrate  and  fat,  acid  substances  are  formed.  Normally, 
however,  it  is  only  protein  metabolism  which  contributes 
appreciable  quantities  of  acid  compounds  for  elimination  in  the 
urine,  the  acid  substances  incident  to  carbohydrate  and  fat 
metabolism  representing  for  the  most  part  intermediary  changes, 
which  eventually  give  rise  to  the  end  products,  carbon  dioxide 
and  water.  When,  on  the  other  hand,  combustion  of  these 
materials  becomes  incomplete,  the  acid  compounds  of  inter- 
mediary metabolism  accumulate  in  the  blood  and  are  excreted 
in  the  urine.  To  this  condition  Naunyn  has  applied  the  term 
"  acidosis."1 

The  acid  substances  or  '-  acetone  bodies  "  with  which  we  are 
particularly  concerned  are  acetone,  diacetic  acid  and  /3-hydroxy- 
butyric  acid.  Protein  or  more  specifically  certain  amino  acids2 
have  been  known  to  yield  acetone  bodies,  but  these  substances 
are  undoubtedly  chiefly  derived  from  incomplete  fat  combustion 
and  hence  a  brief  resume  of  our  conception  of  fat  metabolism  fol- 
lows. 

The  changes  taking  place  in  ingested  fat  prior  to  its  storage 
in  the  tissues  have  been  described  in  Chapter  II.  As  a  prelimi- 
nary to  oxidation  stored  fat"  undergoes  a  cleavage  into  glycerol 
and  fatty  acid.  The  glycerol  is  converted  to  glucose  which  is 
metabolized  in  the  usual  way.  The  fatty  acid  fraction  suffers 
repeated  oxidation  at  the  jS -carbon  atom  until  butyric  acid  is 
formed,  from  this  stage  on  a  somewhat  different  type  of  reaction 
taking  place.  From  butyric  acid  is  formed  /3-hydroxy butyric 
acid  which  is  then  further  oxidized  to  diacetic  acid,  the  latter 
then  through  the  stages  of  acetic  and  formic  acids  finally  giving 

1 .  For  a  general  and  more  extended  discussion  of  acidosis,  see 
A.  E.  Taylor:  "  Digestion  and  Metabolism  "  Philadelphia  and  New  York, 
1912.  For  an  excellent  review  of  the  literature,  reference  may  be  made 
to  James  Ewing:    Arch.  Int.  Med.,  1908,  II,  pp.  330,  448. 

2.  See  page  70. 

79 


80  PATHOLOGICAL  CHEMISTRY 

rise  to  the  end  products,  carbon  dioxide  and  water.  Associated 
with  this  process  there  is  a  side  reaction*  whereby  diacetic  acid 
is  converted  into  acetone,  although  normally  this  occurs  to  but 
a  slight  extent,  only  the  merest  traces  of  acetone  appearing  in  the 
urine.1  However,  when  fat  combustion  suddenly  becomes 
excessive,  this  side  reaction  is  accentuated,  the  oxidation  of 
diacetic  acid  and  /3-hydroxybutyric  acid  is  incomplete,  acetone 
bodies  appear  in  the  urine,  and  we  have  the  condition,  acidosis. 
An  excessive  combustion  of  fat  becomes  necessary  for  the  proper 
maintenance  of  heat  production  whenever  there  is  a  paucity  of 
carbohydrate,  which  is  apparently  more  readily  oxidized  than 
fat.  Thus  starvation  and  the  early  period  of  a  protein-fat  diet 
are  attended  with  acidosis,  which  quickly  disappears  after  the 
administration  of  carbohydrate,  and  which  in  the  case  of  the  pro- 
tein-fat diet  may  vanish  spontaneously,  the  fat  burning  mechan- 
ism presumably  having  improved  with  use ;  or  it  may  be  that 
there  is  a  better  utilization  of  the  carbohydrate  of  protein  origin. 
In  the  mild  stages  of  diabetes  where  total  withdrawal  of 
carbohydrate  is  recommended,  a  marked  elimination  of  acetone 
bodies  is  noted.  However,  as  von  Noorden2  points  out,  this  is 
transient  and  a  physiological  phenomenon,  being  observed  even 
in  normal  individuals  under  similar  conditions  of  diet.  In  the 
advanced  stages  of  diabetes  and  in  certain  other  conditions  there 
is  probably  a  specific  defect  in  fat  -combustion,  and  the  elimina- 
tion of  acetone  bodies  is  but  little  influenced  by  the  utilization  of 
carbohydrate.  If  the  individual  suffering  with  severe  diabetes 
could  utilize  fat  in  the  normal  manner,  his  condition  might  be 
greatly  alleviated,  but  the  defect  in  this  process  removes  a  very 
efficient  source  of  heat,  and  gives  rise  to  substances  which  con- 
tribute to  the  usually  fatal  outcome.  The  acidosis  of  diabetes 
diminishes  whenever  the  carbohydrate  tolerance  improves,  the 
change  being  probably  due  to  the  generally  improved  condition 
which  is  shared  by  the  fat -burning  mechanism.  At  any  rate 
such  a  view  is  more  acceptable  than  the  rather  vague  expression 
of  Rosenfeld  that  fat  can  burn  only  "  in  the  flame  of  carbo- 
hydrates." 

1  There  is  said  to  be  a  daily  elimination  of  0.01  gram  of  acetone 
from  normal  individuals. 

2  .  For  valuable  suggestions  on  dietetic  and  alkali  therapy  in  acidosis, 
reference  may  be  made  to  von  Noorden's  "  New  Aspects  of  Diabetes," 
New  York,  1912. 


ACIDOSIS  81 

In  severe  diabetes  it  is  probable  that  the  oxidation  of  the  fatty 
acid  is  normal  down  to  the  butyric  acid  stage.  From  here  on 
the  combustion  may  be  incomplete  or  defective.  The  early 
period  of  the  acidosis  may  be  attended  with  merely  a  moderately 
strong  acetonuria,  although  as  the  condition  becomes  more 
severe  the  precursors  of  acetone,  viz.:  diacetic  and  /3-hydroxy- 
butyric  acids,  likewise  appear  in  the  urine.  In  still  later  stages 
of  the  disease,  oxidation  of  /3-hydroxybutyric  acid  may  be 
markedly  diminished,  in  which  case  this  substance  appears  in  the 
urine  in  large  amounts  associated  with  only  nominal  quantities 
of  diacetic  acid  and  acetone.  In  acidosis,  the  elimination  of  10 
to  20  grams  of  acetone  bodies  is  not  uncommon,  and  figures  as 
high  as  50  or  even  100  grams  have  been  recorded. 

The  degree  of  acidosis  in  a  diabetic  on  a  protein-fat  diet  is 
influenced  somewhat  by  the  nature  of  the  fatty  acid  fraction  of 
the  fat.  For  example,  one  molecule  of  stearic  and  palmitic  acids 
each  yields  one  molecule  of  butyric  acid,  while  one  molecule  of 
oleic  acid  gives  rise  to  more  than  one  molecule  of  butyric  acid. 
Thus  fat  mixtures  fluid  at  room  temperature — those  containing  a 
preponderance  of  olein,  such  as  olive  oil — yield  larger  quantities 
of  acetone  bodies  than  do  the  more  solid  fats,  e.g.,  the  fats  of 
beef  and  mutton.  Butter  which  contains  preformed  butyric  acid 
esters  also  tends  to  accentuate  the  condition  of  acidosis.  Never- 
theless it  may  be  necessary  to  include  butter  and  olive  oil  in  the 
diet,  as  the  continued  use  of  beef  or  mutton  fats  is  borne 
with  difficulty. 

It  has  been  stated  that  the  fatty  acids  are  oxidized  to  carbon 
dioxide  and  water  through  the  stages  of  butyric  acid,  diacetic 
acid,  etc.  This  applies  only  to  fatty  acids  with  an  even  number  of 
carbon  atoms,  such  as  the  ordinary  fatty  acids  of  our  dietary, 
stearic,  palmitic  and  oleic  acids.  Fatty  acids  with  an  uneven 
number  of  carbon  atoms  cannot  be  transformed  into  butyric 
acid,  but  according  to  the  recent  studies  of  Ringer1  they  may  be 
transformed  into  dextrose  in  so  far  as  they  are  convertible  into 
propionic  acid.  The  oxidation  of  the  higher  fatty  acids  of  this 
type  to  the  propionic  acid  stage  would  thus  furnish  energy  to 
the  diabetic  without  contributing  to  the  acidosis;  on  the  contrary, 
there  would  be  an  antiketogenic  influence. 

The  relation  of  the  acetone  bodies  to  coma  is  as  yet  not 
definitely  understood,  and  it  can  hardly  be  discussed  at  length 

1.    Ringer:     Jour.  Biol.    Chetn.,  1913,  XIV,  p.  43. 


82  PATHOLOGICAL  CHEMISTRY 

in  the  present  brief  chapter.  We  can  only  point  out  that  the 
toxic  action  is  not  due  entirely  to  the  acid  nature  of  the  acetone 
bodies,  since  their  salts  are  also  somewhat  toxic.  Furthermore, 
susceptibility  to  their  toxic  influence  is  evidently  subject  to  great 
variations  since  large  eliminations  of  acetone  bodies  are  some- 
times encountered  with  no  indication  of  coma,  and  on  the  other 
hand,  coma  may  occur  with  only  a  moderate  acetonuria.  The 
suggestion  has  been  made  that  acidosis  does  not  produce  coma, 
but  that  both  these  conditions  are  results  of  some  common  toxic 
agent.  It  is  not  improbable  that  the  influence  of  acetone  upon 
respiration  may  play  an  important  role.1  The  nephritis  asso- 
ciated with  long-continued  acidosis  may  bear  some  relation  to 
the  development  of  coma. 

The  relatively  large  amounts  of  acid  compounds  in  the  blood 
and  tissues  induce  the  liberation  of  increased  quantities  of  bases, 
principally  ammonia;  and  indeed  the  estimation  of  ammonia 
often  forms  a  simple  and  valuable  guide  in  following  the  progress 
of  a  case  of  acidosis.  Normally,  0.3  to  0.6  gram  of  ammonia  is 
eliminated  in  the  urine  daily.  In  acidosis,  outputs  of  2  to  10 
grams  are  not  uncommon,  and  Magnus-Levy  has  calculated 
that  every  gram  of  ammonia  in  excess  of  the  normal  corresponds 
to  approximately  6  grams 'of  /3-hydroxybutyric  acid.  It  should 
be  noted,  however,  that  the  ammonia  excretion  is  not  an  infallible 
indicator  of  the  degree  of  acidosis,  since  the  quantitative  output 
of  ammonia  is  influenced  by  several  other  factors,  e.g.,  total 
nitrogen  output,  defective  urea  formation  in  the  liver,  etc. 
(See  Chapter  III.)  At  times,  therefore,  the  direct  estimation 
of  the  individual  acetone  bodies  may  be  desirable.  There  is 
little  difficulty  in  obtaining  sufficient  ammonia  for  neutralizing 
the  abnormal  acids,  but  it  should  be  understood  that  in  addition 
to  ammonia,  all  cations  of  the  body  contribute  to  this  neutraliza- 
tion, and  there  is  thus  the  tendency  to  disturb  the  mineral 
equilibrium  in  the  tissues.  In  all  cases  of  marked  acidosis, 
administrations  of  alkalies  are  indicated.  The  alkali — usually 
sodium  carbonate  or  sodium  bicarbonate — serves  merely  to 
facilitate  the  removal  of  the  acid  compounds  but  has  no  important 
influence  upon  their  formation.  The  use  of  alkali  has  frequently 
given  temporary  relief,  probably  owing  to  the  resulting  diuresis 

1.  Cf.  Henderson  and  Underhill:  Amer.  Jour.  Physioi.,  1911, 
XXVIII,  p.  275. 


ACIDOSIS  83 

during  which  considerable  toxic  material  was  removed,  but  the 
alkali  therapy  has  not  given  generally  satisfactory  results. 
Taylor1  has  made  the  interesting  suggestion  that  the  logical 
procedure  would  be  to  administer  a  mixture  of  the  salts  of  sodium 
potassium  and  calcium  rather  than  of  sodium  alone,  as  in  this 
manner  there  would  be  more  opportunity  for  maintaining  mineral 
equilibrium  in  the  tissues. 

Acidosis  may  be  a  concomitant  of  a  number  of  abnormal 
conditions  in  addition  to  diabetes.  <  Its  occurrence  in  starvation 
has  already  been  noted.  Here  the  eliminations  of  acetone 
bodies  may  be  quite  as  high  as  in  the  severe  acidosis  of  diabetes. 
Acidosis  has  been  observed  in  eclampsia  and  pernicious  vomiting 
of  pregnancy,  although  in  these  cases,  and  especially  in  the  latter 
it  is  probably  to  be  attributed  to  the  inadequate  state  of  nutri- 
tion.2 

Characteristic  cyclic  vomiting,  occurring  usually  in  children, 
is  accompanied  by  acidosis.  Recurrence  is  very  common,  each 
attack  as  a  rule  lasting  two  or  three  days,  or  occasionally  as 
long  as  two  weeks.  The  condition  is  apparently  uninfluenced 
by  the  administration  of  carbohydrates.  It  has  been  argued  by 
some  that  we  have  here  a  condition  of  acid  intoxication,  although 
Ewing  is  of  the  opinion  that  we  are  dealing  with  various  dis- 
turbances of  metabolism  including  defective  hepatic  function 
and  poisoning  with  intestinal  putrefactive  products. 

Acidosis  following  anesthesia  is  also  well  recognized,  chloroform 
being  more  potent  in  this  respect  than  ether.  In  some  cases  only 
a  mild  acetonuria  is  observed,  while  in  others,  especially  in 
children,  this  urinary  finding  is  associated  with  nausea,  vomiting 
and  somnolence,  these  symptoms  lasting  only  a  few  days.  In 
still  other  cases  the  condition  may  be  serious  indeed,  involving 
jaundice,  acidosis,  convulsions  and  coma,  usually  terminating 
in  death  in  a  few  days.  In  addition  to  the  acetone  bodies, 
ammonium  lactate  and  increased  amounts  of  amino  acids  appear 
in  the  urine.  The  condition  is  uninfluenced  by  the  presence  or 
absence  of  sugar  in  the  diet,  and  is  attributed  to  necrosis  of  the 
liver  induced  by  the  chloroform.  Subjects  predisposed  to  this 
condition  are  generally  regarded  as  poor  surgical  risks. 

In  typhoid  fever,  scarlatina,  diphtheria,  measles  and  many 
other  infections  a  marked  acidosis  has  been  observed,  although 

1.  A.  E.  Taylor:   hoc.  cit.,  p.  330. 

2.  Cf.  Underhill  and  Rand:    Arch.  Int.  Med.t  1910,  V,  p.  61. 


84  PATHOLOGICAL  CHEMISTRY 

the  degree  of  acidosis  bears  no  striking  parallelism  to  the  severity 
of  the  disease.  Administrations  of  sugar  sometimes  do  and  at 
other  times  do  not  influence  the  course  of  the  acidosis.  These 
instances  of  acidosis  are  attributed  in  part  to  the  element  of 
starvation,  and  in  part  to  the  toxic  destruction  of  tissues.  A 
similar  dual  explanation  is  offered  to  account  for  the  occurrence 
of  acidosis  following  the  use  of  many  drugs,  e.g.,  phosphorus, 
arsenic  and  lead  compounds,  antipyrin,  morphine,  atropine, 
curare  and  carbon  monoxide. 

LABORATORY   PROCEDURES. 

In  clinical  work  it  will  usually  be  found  sufficient  to  employ 
qualitative  tests  for  acetone  and  diacetic  acid  and  when  these 
tests  indicate  the  existence  of  an  acidosis,  to  estimate  the  degree 
of  this  acidosis  indirectly  with  the  simple  formaldehyde  titration 
method  for  ammonia.1  For  acetone,  the  Rothera  test  has 
been  found  very  satisfactory,  while  the  simple  Gerhardt  test 
for  diacetic  acid  is  sufficiently  accurate  in  the  probable  absence 
of  interfering  drugs.  Acetone,  diacetic  acid  and  /3-hydroxy buty- 
ric acid  may  be  directly  estimated  with  great  accuracy  by  the 
Folin,  Folin-Hart  and  Shaffer  methods,  but  time  will  hardly 
allow  this  in  routine  clinical  work. 

1.  Acetone. — 

a.  Rothera1  s  Test.2 — To  5  to  10  cc.  of  urine,  add  about  a  gram 
of  ammonium  sulphate,  2  to  3  drops  of  a  freshly  prepared 
aqueous  solution  of  sodium  nitroprusside,  and  then  2  cc.  of 
strong  ammonium  hydroxide,  which  may  be  stratified  upon  the 
urine.  A  positive  reaction  is  indicated  by  the  slow  development 
of  a  characteristic  permanganate  color.  The  delicacy  is  1  to 
20,000. 

b.  Gunning's  Iodoform  Test. — To  about  5  cc.  of  urine  or 
distillate  in  a  test  tube,  add  a  few  drops  of  iodine-potassium 
iodine  solution  and  enough  ammonia  to  form  a  black  precipitate 
of  nitrogen  iodide.  Allow  the  tube  to  stand  (the  length  of 
time  depending  upon  the  amount  of  acetone  present)  and  note  the 
formation  of  a  yellowish  sediment  consisting  of  iodoform. 

2.  Diacetic  Acid. — 

a.  Gerhardt1  s  Test. — To  about  5  cc.  of  urine,  add  ferric  chloride 
solution,  drop  by  drop,  until  no  more  precipitate  forms.     In 

1.  See  Chapter  III,  p.  43. 

2.  Rothera:     Jour.  Physiol.,  1908,  XXXVII,  p.  491. 


TESTS  FOR  ACETONE  BODIES  85 

the  presence  of  diacetic  acid,  a  Bordeaux-red  color  is  produced. 
This  color  may  be  somewhat  masked  by  the  precipitate  of  ferric 
phosphate,  in  which  case  the  fluid  should  be  filtered.  A  positive 
result  indicates  the  possible  presence  of  diacetic  acid.  A  variety 
of  drugs  or  their  derivatives  when  present  in  the  urine  yield  a 
similar  reaction. 

b.  Arnold-Lipliawsky  Reaction. — This  reaction  is  somewhat 
more  delicate  than  that  of  Gerhardt  and  much  more  specific. 
Five  cc.  of  urine  and  an  equal  volume  of  the  Arnold-Lipliawsky 
reagent1  are  mixed  in  a  test  tube,  a  few  drops  of  ammonium 
hydroxide  added,  and  the  tube  shaken  vigorously.  A  brick 
red  color  will  be  produced.  Ten  to  20  cc.  of  hydrochloric  acid 
(sp.  gr.  1.19)  are  added  to  1  to  2  cc.  of  this  colored  solution, 
then  3  cc.  of  chloroform,  2  to  4  drops  ferric  chloride  and  the 
fluid  carefully  mixed  without  shaking.  Diacetic  acid  is  indicated 
by  the  chloroform  assuming  a  violet  or  blue  color.  If  diacetic 
acid  is  absent,  the  color  may  be  yellow  or  light  red. 

3 .  fi-hydroxybutyric  Acid. — To  20  cc.  of  urine,  in  an  evaporat- 
ing dish  add  2  drops  of  acetic  acid,  and  concentrate  at  a  gentle 
heat  until  the  volume  is  reduced  to  ^about  5  cc.  Acidify  the 
residue  with  a  few  drops  of  concentrated  hydrochloric  acid,  add 
plaster  of  Paris  to  make  a  thick  paste,  and  allow  to  partially 
"  set."  Then  break  up  with  a  stirring  rod  and  extract  twice 
with  ether  by  stirring  and  decantation.  Pour  off  ether  and  evapo- 
rate spontaneously  or  over  the  water  bath.  Dissolve  the  residue 
in  water,  filter,  and  divide  between  two  test  tubes.  To  one  tube, 
add  1  cc.  hydrogen  peroxide,  and  warm  gently  for  about  one 
minute,  and  then  allow  to  cool.  Add  to  each  test  tube 
about  one  gram  of  ammonium  sulphate,  a  few  drops  of  a  freshly 
prepared  water  solution  of  sodium  nitroprusside  and  overlay  the 
solutions  with  2  cc.  of  concentrated  ammonium  hydroxide  as 
in  the  Rothera  test  for  acetone.  Allow  the  tubes  to  stand 
for  four  hours.  At  the  end  of  this  time  compare.  The  tube  to 
which  the  peroxide  was  added  will  show  a  purplish  red  contact 
if  /3-hydroxy butyric  acid  was  originally  present.      If  both  tubes 

1 .  The  Arnold-Lipliawsky  reagent  consists  of  two  separate  solutions 
"  a  "  and  "  b  "  which  are  mixed  in  the  ratio  of  1  :  2  just  prior  to  use. 
"  a  "  is  one  per  cent,  solution  of  potassium  nitrite  and  M  b  "  is  one  per 
cent,  aqueous  solution  of  £-amino-acetophenon,  to  which  just  enough 
hydrochloric  acid  has  been  added  (about  2  cc.)  drop  by  drop,  to  cause  the 
solution,  which  is  at  first  yellow,  to  become  entirely  colorless. 


86  PATHOLOGICAL  CHEMISTRY 

p 

are  now  shaken,  the  difference  in  color  will  be  seen  throughout  the 
fluid.    Albumin,  if  present,  should  be  removed. 

4.  Determination  of  Acetone  and  Diacetic  Acid.  Folin-Hart 
Method. — The  same  type  of  apparatus  is  here  employed  as  used 
in  the  estimation  of  ammonia  by  the  Folin  method,1  except 
that  in  the  place  of  the  aerometer  cylinder,  a  large  test  tube 
about  two  inches  in  diameter  is  employed.  The  method: 
An  excess  of  N/10  iodine  solution  and  an  excess  of  40  per  cent, 
potassium  hydroxide  are  accurately  measured  into  a  wide-mouthed 
bottle  containing  200  cc.  of  water.  An  aerometer  cylinder 
containing  alkaline  hypoiodite  solution  is  arranged  to  absorb 
any  acetone  which  may  be  present  in  the  air  of  the  labora- 
tory, and  between  the  cylinder  and  the  bottle  the  large  test  tube 
is  suspended.  This  test  tube  should  contain  20  cc.  of  the  urine 
to  be  examined,  10  drops  of  10  per  cent,  phosphoric  acid,  10 
grams  of  sodium  chloride,  and  a  little  petroleum.  It  should  be 
raised  sufficiently  high  to  facilitate  the  easy  application  of  heat. 
Otherwise,  the  arrangement  is  the  same  as  for  ammonia,  except 
that  the  air  current  is  passed  more  slowly  (and  only  for  25  min- 
utes) to  remove  acetone.  At  this  point  the  absorption  bottle  is 
removed  and  another  substituted.  The  contents  of  the  large 
test  tube  are  now  heated  just  to  the  boiling  point  and  after  a  five 
minute  interval  again  heated.  At  the  end  of  the  second  twenty- 
five  minutes,  the  diacetic  acid  will  have  been  transformed  to 
acetone,  removed  to  the  absorption  bottle  and  there  held  as 
iodoform. 

Titrate  the  excess  of  iodine  in  both  absorption  bottles  until  a 
light  yellow  color  is  observed.  At  this  point  a  few  centimeters 
of  starch  paste  should  be  added,  and  the  mixture  titrated  until 
no  blue  color  is  visible. 

One  cc.  of  N/10  iodine  is  equivalent  to  0.967  milligram  of 
acetone.      The    diacetic    acid    may    be    conveniently   recorded 
in  terms  of  acetone. 
5.  Determination  of  fi-hydroxybutyric  Acid. — 

For  the  most  accurate  purposes,  the  method  of  Shaffer,  in 
which  the  /3-hydroxybutyric  acid  is  oxidized  to  acetone  and 
ultimately  titrated  as  above,  is  perhaps  the  method  of  choice. 
For  practical  purposes,  however,  the  method  of  Black  is  more 
easily  carried  out  and  is  likewise  accurate.    The  method:    Fifty 


1.   See  Chapter  III,  p.  44. 


ESTIMATION  OF  ACETONE  BODIES  87 

cc.  of  urine  are  rendered  faintly  alkaline  with  sodium  carbonate, 
evaporated  to  one-third  the  original  volume,  and  finally  con- 
centrated to  about  10  cc.  over  a  water  bath.  The  residue  is 
cooled,  made  distinctly  acid  with  hydrochloric  acid  and  enough 
plaster  of  Paris  added  to  form  a  thick  paste.  When  the  mass 
has  begun  to  "set,"  reduce  it  to  the  consistency  of  coarse  meal 
with  a  heavy  glass  rod,  and  extract  in  a  Soxhlet  apparatus  with 
ether  for  two  hours.  Now  evaporate  the  ether  in  the  air,  dissolve 
the  residue  in  water,-  filter  (adding  a  little  bone-black  if  neces- 
sary to  obtain  a  clear  filtrate)  and  make  up  to  a  known  volume 
with  water  (25  cc.  or  less).  Ascertain  the  amount  of  /3-hydroxy- 
butyric  acid  in  the  50  cc.  of  urine  by  its  rotation.  The  specific 
rotation  of  /3-hydroxybutyric  acid  is  —  24. 12°  for  solutions  of  1 
to  11  per  cent. 


CHAPTER  VII. 

PIGMENTURIA. 

A  variety  of  pigments  and  pigment-forming  substances  appear 
in  the  urine  under  both  normal  and  pathological  conditions. 
Those  which  merit  consideration  are  urochrome,  urobilin,  uro- 
erythrin,  bile  pigments,  hematoporphyrin,  hemoglobin,  methem- 
oglobin,  and  melanin,  all  of  which,  except  the  last,  having  a 
common  origin,  viz.,  the  blood  pigment.  Certain  substances  as 
homogentisic  acid,  hydrcchirxn  ard  catechol,  "when  present 
render  the  urine  dark  upon  standing,  while  under  appropriate 
conditions  certain  other  substances  develop  pigments,  or  yield 
color  reactions.  Among  such,  may  be  mentioned  the  formation 
of  indigo  blue  from  indican  under  the  action  of  oxidizing  reagents, 
the  formation  of  a  red  color  by  indoleacetic  acid  (urorosein)  with 
hydrochloric  acid  in  the  presence  of  nitrites,  Ehrlich's  diazo 
reaction,  and  Ehrlich's  aldehyde  reaction,  which  is  probably 
referable  to  the  presence  of  urobilinogen. 

Urochrome. — The  yellow  color  of  normal  urine  is  probably 
dependent  upon  several  pigments,  but  in  greatest  part  upon 
urochrome.  Urochrome  is  apparently  closely  related  to  uro- 
bilin, since  the  latter  may  be  readily  converted  into  urochrome 
through  evaporation  of  its  aqueous  ether  solution. 

Urobilin. — When  the  excreted  urine  is  exposed  to  the  action 
of  light,  it  is  regularly  found  to  contain  a  yellow  pigment,  urobilin, 
which  is  derived  from  a  chromogen,  urobilinogen,  under  the 
action  of  light  and  air.  It  is,  however,  claimed  that  no  urobilin 
is  present  in  freshly  voided  normal  urine.  Urobilin  is  probably 
similar  to,  if  not  identical  with,  the  hydrobilirubin  of  the  feces. 
Certain  investigators  have  claimed  that  there  are  various  forms 
of  urobilin,  e.g.,  normal,  febrile,  etc.  Urobilin  is  increased  in 
most  febrile  diseases,  erysipelas,  malaria,  pneumonia,  scarlet 
fever,  etc.,  also  in  cirrhosis  of  the  liver,  carcinonia  of  the  liver, 
catarrhal  icterus,  pernicious  anemia,  appendicitis,  etc.  In 
general,  disturbances  of  the  liver  and  excessive  destruction  of 
red  blood  cells  favor  its  increase.  Poisoning  with  such  drugs 
as  acetanilid,  which  brings  about  a  destruction  of  red  cells, 

88 


PIGMENTURIA  89 

may  cause  the  elimination  of  enough  urobilin  to  produce  a 
urine  almost  black  in  color  as  has  been  found  in  a  specimen 
examined  by  one  of  us1. 

Uroery thrin. — It  is  to  this  pigment  that  the  beautiful  red 
color  often  found  in  urinary  sediments,  especially  urate  sedi- 
ments, is  generally  due.  It  frequently  occurs,  although  in  very 
small  amounts,  dissolved  in  normal  urine.  After  great  muscular 
activity,  digestive  disturbances,  fevers,  circulatory  disturbances 
of  the  liver,  and  in  many  other  pathological  conditions,  the 
elimination  of  Uroery  thrin  is  found  to  be  increased. 

Bile  Pigments. — Bile  pigments  are  not  normally  found  in 
urine,  and  when  present  may  be  regarded  as  a  symptom  of 
disease.  Of  the  bile  pigments,  bilirubin  alone  is  encountered  in 
freshly  voided  urine;  other  pigments,  biliverdin,  etc.  being 
formed  as  oxidation  products  of  the  bilirubin  on  standing. 
Urine  containing  bile  is  yellowish -green  to  brown  and  upon  shak- 
ing, the  foam  takes  on  a  bright  yellow  color.  Normally,  bilirubin, 
which  is  formed  from  the  blood  pigment  in  the  liver,  is  eliminated 
into  the  small  intestine  and  there  transformed  to  hydrobilirubin 
and  excreted  in  the  feces,  while  a  certain  portion  after  being 
reabsorbed  into  the  blood  is  eliminated  in  the  urine  in  the  form 
of  one  of  the  normal  urinary  pigments.  Whenever,  for  any  cause, 
the  outflow  of  bile  is  impeded,  bilirubin  is  absorbed  by  the 
lymphatics  and  eliminated  in  the  urine.  This  may  be  brought 
about  by  obstruction  of  the  bile  ducts,  especially  the  common 
duct,  due  to  simple  swelling  of  its  mucous  membrane,  as  in 
catarrhal  jaundice,  to  the  presence  of  a  biliary  calculus,  or  to 
pressure  by  tumors  in  the  duct  or  surrounding  glands.  Choluria 
also  results  in  conditions  where  the  blood  pressure  in  the  liver 
is  lowered,  hepatogenic  icterus;  also  to  an  inability  of  the  liver 
to  transform  the  blood  pigment  to  bile  pigments  as  fast  as  it  is 
brought  to  it,  as  in  cases  of  acute  yellow  atrophy,  pernicious 
anemia,  etc.,  and  is  termed  hematogenic  icterus. 

Hematoporphyrin. — Hematoporphyrin,  a  substance  closely 
related  to  bilirubin,  possibly  an  isomer,  is  occasionally  found  in 
the  urine  in  various  diseases,  more  commonly  after  the  use  of 
certain  drugs,  such  as  quinine,  trional,  and  especially  in  sulfonal 
intoxication.  In  these  conditions,  the  urine  possesses  a  slightly 
reddish  tint,  although  after  sulfonal  it  may  be  more  or  less  deep 

1 .    See  Gordinier:    Boston  Med.  Surg.  Jour.,  1911,  CLXV,  p.  202. 


90  PATHOLOGICAL  CHEMISTRY 

red.  Here  the  color  depends  in  greatest  part,  not  upon  the 
hematoporphyrin,  but  probably  upon  other  reddish  brown 
pigments. 

Blood  and  Blood  Pigments. — When  urine  contains  blood  from 
hemorrhage  of  the  kidney,  or  other  parts  of  the  urinary 
tract,  and  the  formed  elements  of  the  blood  are  found  in  the 
sediment,  the  condition  is  termed  hematuria.  In  such  conditions, 
when  the  quantity  of  blood  is  moderately  large,  the  color  of  the 
urine  may  vary  from  a  red  to  a  dark  brown.  Where  the  hemor- 
rhage is  recent  and  the  urine  fresh,  the  color  is  a  brighter  red. 
In  certain  other  conditions,  however,  the  urine  contains  no  red 
corpuscles,  but  only  the  blood  pigment,  hemoglobin,  or  as  is 
often  the  case  here,  methemoglobin,  which  condition  is  termed 
hemoglobinuria.  Two  factors  may  play  a  part  in  bringing  about 
hemoglobinuria,  viz.,  hemolysis  of  the  red  blood  cells  and  an 
inefficiency  of  the  liver.  The  condition  is  observed  after  poison- 
ing with  chlorates  and  other  drugs,  after  severe  burns,  and  also 
in  the  periodic  appearance  of  hemoglobinuria  with  fever. 

Melanins. — In  certain  pathological  conditions,  viz.,  in  the 
presence  of  melanotic  tumors,  dark  pigments  are  sometimes 
eliminated  with  the  urine.  The  freshly  passed  urine  is  generally 
clear,  and  upon  standing,  darkens  and  may  become  a  very  dark 
brown,  or  even  black,  indicating  that  the  pigment  is  probably 
present  in  the  form  of  a  chromogen  and  is  oxidized  upon  coming 
in  contact  with  the  air. 

Indican. — By  the  action  of  putrefactive  bacteria  in  intestines 
upon  the  amino  acid,  tryptophane,  indole  is  set  free.  This 
indole  is  in  large  part  absorbed  by  the  blood,  carried  to  the  liver 
and  there,  after  oxidation  to  indoxyl,  combined  with  sulphuric 
acid  and  potassium  to  form  indoxyl  potassium  sulphate  or 
indican,  which  subsequently  appears  in  the  urine.  Upon  oxida- 
tion, indican  yields  indigo  blue.  It  has  conclusively  been  shown 
that  indole  is  formed  in  the  body  by  bacterial  action  only, 
although  this  action  is  not  necessarily  confined  to  the  intestine, 
an  increased  indicanuria  being  observed  in  cases  of  empyema, 
putrid  bronchitis,  gangrene  of  the  lung,  etc.  Normally,  5  to 
20  milligrams  of  indican  are  eliminated  in  the  course  of  24  hours, 
but  the  amount  may  be  enormously  increased  in  conditions  of 
excessive  intestinal  putrefaction.  The  hydrochloric  acid  of  the 
gastric  juice  appears  to  regulate,  to  a  certain  extent,  the  degree 


I     PIGMENTURIA  91 

of  this  intestinal  putrefaction,  as  in  cases  of  anachlorhydria 
and  hypochlorhydria,  the  amount  of  indican  may  be  very 
greatly  increased.  We  have  found  that  in  certain  cases  of 
pellagra1  accompanied  by  anachlorhydria  that  the  indican  was 
enormously  increased  (250  mgms.  per  day  in  one  case),  while 
in  those  cases  in  which  free  hydrochloric  acid  was  present,  the 
indicanuria  was  much  less  marked.  Another  factor  in  this 
condition  appeared  to  be  the  lack  of  pepsin.  In  cases  where 
peristaltic  movements  have  been  impeded  as  in  ileus  and  peri- 
tonitis, an  increase  in  indican  generally  occurs,  although  this  is 
hardly  the  case  in  simple  uncomplicated  constipation. 

Indole- Acetic  Acid  (Urorosein). — Under  certain  bacterial 
conditions  in  the  alimentary  tract  in  various  diseases,  especially 
in  cachectic  conditions,  a  substance  has  been  found  to  appear  in 
the  urine  which  becomes  red  when  acidified  with  a  mineral  acid 
(hydrochloric)  in  the  presence  of  an  oxidizing  agent  (nitrites). 
This  red  pigment,  previously  called  urorosein,  has  been  shown 
by  Herter  to  be  indole-acetic  acid. 

Ehrlich's  Diazo  Reaction. — A  chromogen  sometimes  appears 
in  the  urine  under  pathological  conditions,  especially  in  typhoid 
fever,  which,  when  treated  with  diazobenzene-sulphonic  acid 
and  ammonia,  produces  a  characteristic  red  color,  visible  in  the 
foam  on  shaking.  A  positive  reaction  is  obtained  in  the  first 
two  weeks  of  typhoid  fever  in  about  75  per  cent,  of  the  cases. 
It  usually  disappears  at  the  end  of  the  third  week  of  the  disease, 
but  generally  reappears  in  a  relapse.  The  intensity  of  the 
reaction,  as  a  rule,  runs  parallel  with  the  severity  of  the  infec- 
tion. The  diagnostic  import  of  the  reaction  in  typhoid  fever  is 
lessened,  however,  by  the  fact  that  distinct  reactions  are  fre- 
quently obtained  in  measles,  scarlet  fever,  acute  miliary  tuber- 
culosis, pneumonia,  erysipelas,  pyemia,  etc. 

Ehrlich's  Aldehyde  Reaction— It  has  been  shown  by  Ehrlich 
that  under  various  pathological  conditions,  a  deep  cherry-red 
color  will  develop  on  shaking  a  specimen  of  urine  with  a  few 
drops  of  an  acid  solution  of  ^-dimethylaminobenzaldehyde,  and 
that  the  resulting  pigment  can  in  part  be  extracted  with  chloro- 
form. The  reaction,  according  to  O.  Neubauer,  is  due  to  uro- 
bilinogen. Herter  has  also  shown  that  the  administration  of 
skatole  causes  an  intensification  of  the  reaction.  A  positive 
reaction  is  commonly  obtained  in  cases  of  tuberculosis,  but  more 

1.    Myers   and  Fine:    Amer.  Jour.  Med.  Sci.,  1913,  CXLV,  p.  705. 


92  PATHOLOGICAL  CHEMISTRY 

especially  in  conditions  accompanied  by  a  derangement  of  the 
liver  cells,  in  which  condition  it  is  claimed  by  some  clinicians 
to  be  of  considerable  diagnostic  importance. 

LABORATORY    PROCEDURES.. 

In  the  examination  of  the  various  urinary  pigments  mentioned 
above,  the  spectroscope  is  of  very  great  service.  In  fact,  the 
identification  of  the  specific  absorption  bands  may  be  almost 
indispensable  in  certain  cases.  So  far  as  possible,  however, 
simple  chemical  tests  have  been  outlined  below. 

1.  Urobilin. — About  20  cc.  of  the  urine  under  examination 
are  acidified  with  a  few  drops  of  hydrochloric  acid  and  shaken 
gently  with  5  cc.  of  amyl  alcohol.  The  amyl  alcohol  extracts 
the  pigment,  and  when  examined  spectroscopically  will  show 
the  characteristic  urobilin  absorption  bands  between  b  and  F 
(the  green  and  blue  parts  of  the  spectrum).  Upon  treating  the 
amyl  alcohol  extract  with  an  alcoholic  solution  of  zinc  chloride 
and  ammonia,  it  will  show  a  bright  green  fluorescence,  and  appear 
a  faint  pink  color  by  transmitted  light. 

For  a  test  for  urobilinogen,  see  under  Ehrlich's  aldehyde  reac- 
tion. 

2.  Bile  Pigments. — Urine  containing  bile  generally  has  a 
dark  yellow  color  with  a  foam  of  bright  yellow,  which  is  valuable 
evidence  as  to  the  presence  of  bile. 

a.  Gmelin's  Test. — This  test  consists  simply  in  the  careful 
stratification  of  urine  upon  concentrated  nitric  acid  as  in  Heller's 
test  for  albumin,  when,  in  the  presence  of  bile  pigments,  various 
colored  rings  (green,  blue,  violet,  red,  and  yellowish-red)  will  be 
noted  at  the  point  of  contact. 

b.  Smith's  Test. — If  an  alcoholic  solution  of  iodine,  having  a 
good  yellow  color,  is  layered  over  urine  containing  bile,  a  green 
ring  will  be  formed  at  the  point  of  juncture  in  the  presence  of 
bilirubin. 

3.  Hemato porphyrin. — Hematoporphyrin  is  best  identified 
spectroscopically.  Caustic  alkali  of  10  per  cent,  concentration 
is  added  to  an  appropriate  volume  of  urine  in  the  proportion  of 
20  cc.  to  100  cc.  of  urine.  The  hematoporphyrin,  which  is  pre- 
cipitated with  the  earthy  phosphates,  is  filtered  off,  washed, 
transferred  to  a  flask  and  warmed  with  a  small  amount  of  alcohol 
acidified  with  hydrochloric  acid.  Upon  filtering,  the  solution  will 
show  the  absorption  bands  of  hematoporphyrin  in  acid  solution. 


TESTS  FOR  URINARY  PIGMENTS  93 

4 .  Blood  Pigments. — In  conditions  of  hematuria,  chemical  tests 
for  blood  are  usually  superfluous,  inasmuch  as  red  blood  cells 
can  generally  be  detected  in  the  urinary  sediment  upon  micro- 
scopical examination.  In  hemoglobinuria,  no  erythrocytes  are 
present,  and  it  may  be  necessary  to  resort  to  a  spectroscopic 
examination  or  certain  chemical  tests  to  ascertain  the  cause  of 
the  red  to  brown  color  which  the  specimen  of  urine  under  exam- 
ination may  possess.  The  spectroscope  is  particularly  valuable 
here,  because  the  absorption  bands  obtained  from  a  direct 
examination  of  the  filtered  urine  may  enable  one  to  decide  at 
once  whether  the  pigment  is  hemoglobin  or  met  hemoglobin,  or 
to  identify  it  as  e.g.,  hematoporphyrin  without  further  examina- 
tion. When  it  is  simply  necessary  to  identify  the  color  as 
due  to  a  blood  pigment,  the  tests  for  "  occult  "  blood  may  be 
applied  in  a  similar  manner  to  the  directions  given  in  Chapter  II, 
p.  23.  The  guaiac  test,  for  example,  may  be  applied  by  simply 
adding  10  drops  of  the  alcoholic  solution  of  guaiac  to  5  cc.  of  the 
urine  and  then  2  cc.  of  ozonized  turpentine  or  hydrogen  peroxide 
and  allowing  the  tube  to  stand  for  two  to  three  minutes.  In 
the  presence  of  blood,  a  blue  color  will  develop. 

5.  Melanin. — In  the  presence  of  a  melanin  or  melanogen, 
urine  will  show  a  black  precipitate  with  ferric  chloride  or  barium 
hydrate.  Bromine  water  will  also  produce  a  precipitate  which 
is  yellow  at  first,  but  turns  black  on  further  standing.  Care 
should  be  taken  not  to  confuse  melanin  with  an  excess  of  indican. 

6.  Indican. — 

a.  Qualitative  Test. — About  10  cc.  of  faintly  acid  urine  are 
shaken  with  a  small  amount  (0.1  gram)  of  basic  lead  acetate, 
filtered  and  the  clear  filtrate  mixed  with  an  equal  volume  of 
Obermayer's  reagent1  and  about  5  cc.  of  chloroform.  Upon 
shaking,  the  chloroform  will  assume  a  blue  color  if  indican  be 
present,  the  intensity  of  which  will  vary  with  the  amount  of 
indigo  blue  which  has  been  brought  into  solution  by  the  chloro- 
form. Qualitatively  the  depth  of  color  may  be  taken  as  indica- 
ting the  degree  of  indicanuria.  Normally,  only  a  faint  blue  color 
is  produced. 

b.  Quantitative  Estimation. — The  above  qualitative  test  may 
be  made  the  basis  of  a  quantitative  colorimetric  method  for  the 
estimation  of  indican  which  we  have  found  rapid  and  satisfac- 

1 .  Obermayer's  reagent  is  prepared  by  adding  2  to  4  grams  of  ferric 
chloride  to  1  liter  of  concentrated  hydrochloric  acid. 


94  PATHOLOGICAL  CHEMISTRY 

tory.  The  quantity  of  urine  to  be  employed  depends  upon  the 
amount  of  indican  present.  Where  qualitative  tests  have  shown 
a  strong  reaction  for  indican,  it  will  be  found  convenient  to 
employ  35  to  45  cc.  of  urine.  A  little  basic  lead  acetate  is  added 
to  the  faintly  acid  urine,  the  mixture  shaken,  filtered,  and  two 
15  cc.  or  20  cc.  portions  taken  to  serve  as  duplicates,  and  treated 
in  separatory  funnels  with  equal  volumes  of  Obermayer's  reagent, 
and  an  accurately  measured  amount  of  chloroform  (10  cc). 
The  mixture  is  then  shaken,  and  after  the  separation  of  chloro- 
form, this  is  withdrawn,  and  fresh  portions  of  measured  amounts 
of  chloroform  added  until  the  extracts  become  practically  color- 
less. These  extracts  are  then  combined  and  filtered.  The  clear 
filtrate  is  compared  in  a  Duboscq  colorimeter  with  a  standard 
solution  of  pure  indigo  blue1  in  chloroform.  Results  obtained 
by  this  method  have  been  found  to  agree  well  with  those  obtained 
with  the  method  of  Ellinger.     Only  in  a  few  instances  out  of 

1 .  The  use  of  a  chloroform  solution  of  indigotin  as  standard  is  to 
be  preferred  to  the  use  of  a  standardized  Fehling's  solution.  Fehling's 
solution  was  first  employed  as  an  empirical  standard  for  comparison  with 
indican  by  Folin  (Amer.  Jour.  Physiol.,  1905,  XIII,  p.  53)  and  it  was 
our  original  intention  to  ascertain  the  strength  of  this  in  terms  of  indigo 
blue  and  indican.  A  study  of  this  question,  however,  showed  us  that 
proportionate  changes  in  the  depth  of  an  indigotin  solution  did  not  show 
the  same  proportionate  changes  in  matching  up  with  Fehling's  solution. 
Further,  when  Fehling's  solution  was  diluted  one-half  with  water  and 
compared  with  a  concentrated  Fehling's,  it  did  not  show  double  the  colori- 
metric  reading.  On  this  account,  the  use  of  Fehling's  solution  as  a  stand- 
ard was  abandoned.  The  standard  indigotin  was  prepared  by  adding  an 
excess  of  Kahlbaum's  pure  indigotin  to  warm  chloroform,  allowing  it  to 
stand  over  night,  and  then  filtering  to  remove  any  suspended  indigotin. 
The  strength  of  this  standard  was  then  determined  by  titrating  the  indi- 
gotin solution  with  approximately  N/400  potassium  permanganate 
according  to  the  method  of  Ellinger  (Zeitschr.  f.  physiol.  Chetn.,  1903, 
XXXVIII,  p.  178.)  The  method  is  as  follows:  10  cc.  portions  of  the  chlo- 
roform extract  are  evaporated  to  dryness  in  small  extraction  flasks,  treated 
with  10  cc.  of  concentrated  sulphuric  acid  and  heated  on  the  water  bath 
for  five  minutes.  They  are  then  treated  with  100  cc.  of  distilled  water 
and  titrated  with  the  permanganate  until  the  last  trace  of  blue  color 
disappears  and  only  a  pale  yellow  remains.  The  permanganate  is  pre- 
pared by  taking  5  cc.  of  a  solution  of  pure  potassium  permanganate 
(3.0000  grams  to  1  liter)  and  diluting  to  200  cc.  with  water.  One  cc.  of 
this  solution  is  equivalent  to  1 . 5  mgms.  of  indigotin.  To  obtain  the 
value  of  the  indigotin  solution  in  terms  of  indican  multiply  by  1.92. 
This  solution  will  keep  for  a  considerable  length  of  time  in  a  well-stoppered 
bottle  in  the  dark. 


TESTS  FOR  URINARY  PIGMENTS  95 

several  hundred  determinations  has  red  pigment  been  encoun- 
tered which  interfered  with  the  colorimetric  estimation. 

7.  Indole- Acetic  Acid — Urorosein  Reaction. — To  about  10  cc. 
of  urine,  add  2  cc.  of  concentrated  hydrochloric  acid  and  a  few 
drops  of  a  1  per  cent,  solution  of  potassium  nitrite.  In  the 
presence  of  indole-acetic  acid  a  rose-red  color  will  develop. 

8.  Ehrlich's  Diazo  Reaction. — Equal  volumes  of  urine  and  the 
diazo  reagent1  are  thoroughly  mixed  in  a  test  tube  by  shaking 
and  ammonium  hydroxide  quickly  added  in  excess.  The  test  is 
positive  if  the  fluid  assumes  a  deep  cherry-red  color,  and  the 
foam  becomes  a  salmon  pink.  It  should  be  borne  in  mind  that 
the  administration  of  certain  drugs  produces  a  similar  reaction 
(napthalin)  while  others  (tannic  acid,  gallic  acid,  etc.)  diminish 
the  reaction,  or  even  cause  it  to  disappear. 

9.  Ehrlich's  Aldehyde  Reaction. — About  5  cc.  of  urine  are 
treated  with  5  to  10  drops  of  the  reagent,2  agitated  for  a  few 
minutes  and  the  color  noted.  A  positive  reaction  is  shown  by  the 
development  of  a  fine  cherry-red  color,  due  probably  to  urobil- 
inogen. In  case  the  test  is  applied  for  urobilinogen,  it  is  impor- 
tant that  the  urine  sample  should  be  fresh  and  not  long  exposed  to 
light. 

1.  The  diazo  reagent  is  composed  of  two  definite  solutions  which 
are  mixed  just  prior  to  use  in  the  proportions  of  1  part  of  (a)  to  50  parts 
of  (b).  (a)  is  a  0.5  per  cent,  solution  of  sodium  nitrite  and  (b)  is  a  0.5 
per  cent,  solution  of  sulphanilic  acid  in.  5  per  cent,  hydrochloric  acid. 
The  £-aminoacetophenon  solution  employed  for  the  Arnold-Lipliawsky 
diace.tic  acid  test  may  be  substituted  for  the  sulphanilic  acid  solution. 

2.  The  reagent  is  a  solution  of  £-dimethylaminobenzaldehyde  in  a 
mineral  acid.  A  2  per  cent,  solution  in  equal  parts  of  water  and  con- 
centrated hydrochloric  acid  is  convenient  for  this  purpose. 


CHAPTER  VIII. 
Examination  of  Urinary  Sediments. 

The  diagnostic  value  of  the  microscopic  examination  of  urinary 
sediments  is  well  recognized.  In  many  instances  it  serves  not 
only  to  disclose  some  inflammatory  lesion  in  the  urinary  tract, 
but,  taken  together  with  the  clinical  symptoms,  enables  one  to 
determine  the  seat  of  the  lesion,  and  to  follow  the  progress  of  the 
case. 

The  two  general  types  of  sediment  constituents  are  the  or- 
ganized (formed  elements)  and  unorganized  (crystalline  and 
amorphous  material),  the  more  important  of  which  are  included 
in  the  following  lists. 

Organized  Constituents.  Unorganized  Constituents. 

Casts.  Ammonium    magnesium    phos- 

Cylindroids.  phate  ("  triple  phosphate"). 

Leucocytes  (pus  cells).  Calcium  oxalate. 

Erythrocytes.  Calcium  phosphate. 

Epithelial  cells.  Calcium  carbonate. 

Mucous  cylinders.  Uric  acid. 

Spermatozoa.  Sodium  urate   (amorphous  and 

Bacteria,    yeasts,    animal  crystalline), 

parasites,  etc.  Ammonium  urate. 

Hippuric  acid. 
Cystine,   leucine,   tyrosine. 

organized  constituents. 

Casts. — Casts  are  molds  of  the  uriniferous  tubules,  result- 
ing probably  from  the  coagulation  of  an  exudate  within  the 
tubules.  They  vary  considerably  in  length  and  breadth,  depend- 
ing upon  their  place  of  origin  in  the  tubules,  but,  for  the  most 
part,  the  width  of  an  individual  cast  is  uniform  and  the  ends  are 
rounded,  although  tapering  and  twisting  forms  are  occasionally 
observed.  Hyaline  casts  (Fig.  1)  are  made  up  of  a  practically 
uniform  transparent  pale  matrix.  At  times  granules  appear  at 
one  end  or  are  scattered  in  small  numbers  over  the  cast,  giving 

96 


URINARY  SEDIMENTS  97 

rise  to  hyaline- granular  casts.  The  granules  may  completely  cover 
the  hyaline  matrix,  when  the  terms  finely  granular  casts  (Fig.  2) 
or  coarsely  granular  casts  (Fig.  3)  are  used,  depending  upon  the 
size  of  the  granules.  If  morphological  elements  or  fat  droplets 
are  found  adhering  to  the  casts  in  sufficient  numbers,  they  are 
given  corresponding  names,  as  for  example,  leucocytic  (or  pus) 
casts,  blood  casts,  epithelial  casts  and  fatty  casts.  Waxy  casts  (Fig.  2) 
are  light  yellow  and  are  somewhat  larger  and  have  more  clearly 
defined  outlines  than  do  hyaline  casts.  Brown  granular  casts  are 
relatively  short  and  broad  and  appear  to  have  broken  ends.  They 
probably  result  from  disintegration  of  the  epithelial  lining  of  the 
tubules. 

Cylindroids,  False  Casts,  Mucous  Cylinders. — Cylindroids  (Fig. 
6)  resemble  casts  in  structure,  but  are  much  longer  and 
show  tapering  and  branching.  Cast-like  formations  consisting  of 
amorphous  urates  may  occasionally  be  mistaken  for  granu- 
lar casts.  Such  masses,  however,  disappear  on  warming.  Mucous 
cylinders  are  long  tapering  transparent  bodies,  which  are,  as  a 
rule,  much  thinner  than  casts  or  cylindroids. 

The  appearance  of  casts  in  the  urine  cannot  always  be  inter- 
preted as  evidence  of  a  distinct  nephritis.  They  may  occur  in 
the  urine  of  apparently  healthy  individuals  after  cold  baths  or 
severe  muscular  exercise,  just  as  albuminuria  may  follow  these 
activities ;  and  they  may  be  present  in  small  numbers  in  the  urine 
of  many  presumably  normal  people  past  middle  life  who  do  not 
lead  especially  active  lives.  Nevertheless  it  would  seem  reason- 
able to  suppose  that  cylindruria  indicates  some  renal  abnor- 
mality, which,  however,  may  never  be  attended  by  definite 
clinical  symptoms.  It  may  be  that  the  pathological  condition 
is  confined  to  a  few  small  areas  in  the  kidney.  On  the  other 
hand,  post  mortem  examinations  have  frequently  revealed 
pathological  alterations  in  the  kidney,  although  the  urine  had 
been  free  from  casts.  In  chronic  interstitial  nephritis,  casts  may 
be  absent  from  the  urine  for  long  periods  of  time,  while  parenchy- 
matous nephritis  is  quite  regularly  accompanied  by  cylindruria. 
Renal  disturbances  resulting  from  infectious  diseases  cause 
numerous  casts  to  appear  in  the  urine.  The  urine  of  the  first  day 
or  two  after  ether  anesthesia  contains  large  numbers  of  casts, 
which,  however,  rapidly  disappear.  ■  With  regard  to  the  particular 
type  of  cast  observed  in  various  conditions,  it  may  be  mentioned 
that  the  hyaline  and  granular  varieties  occur  in  almost  any  renal 


98  PATHOLOGICAL  CHEMISTRY 

disturbance ;  blood  casts  are  looked  upon  as  characteristic  of  acute 
diffuse  nephritis  and  acute  congestion  of  the  kidney;  fatty  casts 
are  associated  with  fatty  degeneration  of  the  kidney;  and  waxy 
casts  are  characteristic  of  amyloid  disease.  Cylindroids  appear 
in  essentially  the  same  conditions  as  do  hyaline  casts. 

Leucocytes. — Normal  urine  contains  only  a  very  small  number 
of  leucocytes  (Fig.  4),  and  hence  any  noteworthy  increase  may  be 
looked  upon  as  evidence  of  an  inflammatory  process  somewhere  in 
the  urinary  tract,  excepting  in  females  where  the  presence  of  leuco- 
cytes maybe  due  to  contamination  with  vaginal  discharge.  For 
purposes  of  diagnosis,  it  is  desirable  to  learn  the  origin  of  the  leu- 
cocytes. When  of  renal  origin,  they  are  often  associated  with  renal 
epithelial  cells  and  casts.  Catheterization  of  the  ureters  or 
bladder  will  frequently  aid  in  determining  whether  the  pyuria  is 
due  to  pyelitis,  ureteritis,  cystitis,  or  urethritis.  Chronic  nephritis 
is  not  ordinarily  attended  with  marked  pyuria  though  large 
numbers  of  leucocytes  may  be  observed  in  the  acute  condition, 
in  an  acute  exacerbation  of  a  chronic  case,  or  where  there  exists 
a  complicating  inflammatory  process  in  some  other  part  of  the 
urinary  tract.  Pus  appears  in  renal  tuberculosis  in  variable 
amounts — from  only  a  few  leucocytes  to  a  distinct  sediment  of 
pus.  Where  a  pyelitis  is  unilateral,  the  affected  side  may  be 
occluded,  and  so  for  a  time  clear  urine  will  be  obtained.  In  an 
advanced  case  of  cystitis,  where  the  urine  has  become  alkaline, 
the  leucocytes  may  be  decomposed  into  a  mucous-like  mass. 

Erythrocytes. — Erythrocytes  (Fig.  5)  practically  never  appear  in 
normal  urine,  and,  therefore,  when  present,  some  pathological  con- 
dition maybe  presumed  to  exist.  When  only  a  small  number  of 
red  cells  are  present,  the  urine  does  not  present  an  unusual  appear- 
ance, and  their  detection  may  require  the  microscope.  When  blood 
is  present  in  considerable  amount,  the  color  of  the  urine  may  vary 
from  bright  red  to  dark  brown,  depending  upon  the  length  of 
time  the  blood  has  been  in  the  urine.  Here  again  it  is  important 
to  learn  the  source  of  the  hematuria. ,  When  of  renal  origin,  the 
red  cells  are  often  associated  with  casts  and  renal  epithelial  cells ; 
the  blood  is  usually  intimately  mixed  with  the  urine  and  contains 
11  blood  shadows,"  corpuscles  from  which  the  hemoglobin  has 
been  washed  out.  Absence  of  these  associations  will  often  serve 
to  eliminate  the  kidneys  as  the  source  of  the  bleeding.  Renal 
hematuria  may  be  due  to  simple  hyperemia,  renal  tuberculosis, 
nephritis,   infectious  diseases,  and  numerous  other  conditions. 


Fig.  1. — Hyaline  Casts. 


Fig.  2. — Finely  Granular  Casts; 
also  Pus  Cast  and  Waxy  Cast. 


Fig.  3. — Coarsely  Granular  Casts.  Fig.  4. — Pus  Cells  with  Epithelial 

Cells  and  Stringy  Mucus. 


Fig.  5. — Erythrocytes  and 
Leucocytes. 


Fig.  6. — Various  Types  of  Epithelial 
Cells;  also  Cylindroid. 


**> 


\  •  'V 


4      ".  *» 

r5 


•••^. 


Fig.  7. — Ammonium  Magnesium       Fig.  8. — Calcium  Oxalate  Crystals. 
Phosphate  Crystals  with  Amorphous 
Deposit. 


Fig.  9. — Uric  Acid  Crystals. 


4     V  •    •*>. 

i 

Fig.  10. — Amorphous  Urates. 


-         • 


<n 


>  <^  * 


*    *-- 


*. 

*& 


*~* 


4 


>     . 


Fig.  11. — Ammonium  Urate 
Crystals. 


4  K 


'  ■*  "•■'■  ■■•■■  K  -~& 


5 


cvc 


K      ,♦     J> 


Fig.  12. — Calcium  Phosphate  and 
"  Triple  Phosphate  "   Crystals. 


URINARY  SEDIMENTS  99 

Blood  cells  from  the  urethra,  prostate,  or  bladder  usually  present 
a  normal  or  crenated  appearance. 

Epithelial  Cells. — Under  ordinary  conditions  the  urine  contains 
a  small  number  of  epithelial  cells  (Fig.  6),  and  when  there  is  any 
notable  increase,  some  abnormal  condition  is  indicated.  It  should 
be  remembered  that  in  the  female  considerable  numbers  of  pave- 
ment epithelial  cells  from  the  vagina  are  present  in  the  urine, 
if  voided .  Three  types  of  epithelial  cells  may  be  observed  in  urine, 
viz:  round  cells,  caudate  cells  and  large  flat  cells.  Round  cells 
are  slightly  larger  than  pus  cells  and  contain  large,  clearly  defined 
nuclei.  They  are  found  in  the  uriniferous  tubules  and  in  the 
deeper  layers  of  the  mucous  membrane  of  other  parts  of  the 
urinary  tract.  Caudate  cells  are  derived  from  the  superficial 
layers  of  the  kidney  pelvis  and  the  neck  of  the  bladder.  Flat 
cells  may  be  derived  from  the  ureters,  bladder  or  vagina. 
Attempts  have  been  made  to  locate  the  abnormal  condition  by 
the  type  of  cell  appearing  in  the  urine,  but  it  cannot  be  said  that 
any  one  type  of  cell  is  characteristic  of  a  definite  region.  However, 
certain  associations  often  aid  in  diagnosis.  Thus,  when  round 
cells  predominate  and  are  accompanied  by  casts  and  albuminuria, 
the  cells  are  probably  of  renal  origin.  When  casts  and  albu- 
minuria are  absent,  the  simultaneous  presence  of  large  numbers  of 
pus  cells  points  to  the  kidney  pelvis  as  the  origin  of  the  round  cells. 

In  urine  which  has  been  exposed  for  some  time  after  voiding 
or  has  been  infected  while  in  the  bladder,  a  variety  of  bacteria 
may  be  observed.  Yeasts  cells  showing  characteristic  budding 
are  often  noted  in  urine,  especially  in  urine  containing  sugar. 

UNORGANIZED    CONSTITUENTS. 

The  identification  of  the  various  unorganized  sediments  is  of 
much  less  importance  diagnostically  than  that  of  the  organized. 
However,  they  often  aid  in  obtaining  a  picture  of  the  condition, 
and  in  certain  rare  cases,  where,  for  example,  cystine,  leucine  or 
tyrosine  appear  in  the  urine,  considerable  importance  may  be 
attached  to  the  examination. 

The  character  of  the  sediment  is  for  the  most  part  determined 
by  the  reaction  and  concentration  of  the  urine.  Thus,  uric  acid, 
sodium  urate,  and  calcium  oxalate  crystals  are  usually  noted  in 
acid  urines;  while  crystals  of  triple  phosphate,  calcium  phosphate, 
calcium  carbonate  and  ammonium  urate  are  observed  in  alkaline 
urines.    Triple  phosphate  crystals  are  most  abundant  in  alkaline 


100  PATHOLOGICAL  CHEMISTRY 

urines,  although  they  are  not  infrequently  encountered  in  urine 
of  neutral  or  slightly  acid  reaction. 

Simple  inspection  of  the  urinary  deposit  will  often  give  a  clue 
as  to  its  nature.  For  example,  a  granular  brick  red  sediment  is 
referable  to  uric  acid;  a  pink  amorphous  deposit,  dissolving  when 
warmed,  indicates  sodium  urate;  a  white  flocculent  sediment, 
which  disappears  on  the  addition  of  acetic  acid,  points  to  the 
presence  of  phosphates  or  carbonates. 

The  crystallization  of  calcium  oxalate,  uric  acid,  etc.,  does  not 
necessarily  indicate  an  absolute  increase  in  the  elimination  of 
these  substances.  It  simply  means  that  the  conditions,  such  as 
reaction,  concentration  and  temperature,  were  favorable  for 
their  crystallization.  It  is  important  to  bear  in  mind  that  when 
such  favorable  conditions  continually  exist,  there  is  the  possibility 
of  the  formation  of  calculi. 

Ammonium  Magnesium  Phosphate  ("  Triple  Phosphate  ") . — This 
compound  (Fig.  7)  crystallizes  as  prisms  ("  coffin  lids  ")  and  in 
a  feathery  arrangement,  the  former  being  the  more  character- 
istic. They  are  observed  in  urines  which  have  been  exposed  for 
some  time,  and  in  disorders  in  which  there  is  retention  of  urine  in 
the  bladder,  as  in  cystitis  and  enlarged  prostate. 

Calcium  Oxalate. — Calcium  oxalate  (Fig.  8)  appears  in  the  urine 
as  octahedra  of  various  sizes,  and  occasionally  in  a  dumb-bell 
formation.  The  octahedral  variety  may  at  times  be  confused 
with  small  prismatic  crystals  of  triple  phosphate,  and  the  dumb- 
bell type  with  calcium  carbonate  crystals.  However,  triple 
phosphate  and  calcium  carbonate  are  soluble  in  acetic  acid,  while 
calcium  oxalate  is  not.  Calcium  oxalate  crystals  may  be  noted 
after  ingestion  of  tomatoes,  rhubarb,  garlic,  asparagus,  oranges., 
etc. ;  also  in  diabetes  mellitus,  phthisis,  neurasthenia,  etc. 

Calcium  Phosphate. — Calcium  phosphate  (Fig.  12)  occurs  in 
both  amorphous  and  crystalline  forms.  The  crystals  are  relatively 
long  and  wedge-shaped,  often  appearing  in  rosette  arrangements. 
In  this  form  they  may  resemble  sodium  urate  crystals,  but  can 
be  distinguished  from  the  latter  by  their  solubility  in  acetic  acid. 

Calcium  Carbonate. — This  substance  may  crystallize  in  the  form 
of  dumb-bells,  which  are  somewhat  smaller  than  the  dumb-bell 
variety  of  calcium  oxalte  crystals. 

Uric  Acid. — Uric  acid  (Fig.  9)  may  crystallize  in  a  number  of 
forms,  e.g.,  wedge-shape,  oval  with  pointed  ends,  irregular  plates, 
and  in  various  rosette  arrangement  of  these  forms.     As  a  rule, 


URINARY  SEDIMENTS  101 

they  are  colored  brownish-red,  although  perfectly,  colorless 
crystals  may  occasionally  be  observed,  Uric  acid  crystals  may 
appear  in  the  urine  after  profuse  perspiration,  vomiting,  diarrhea, 
and,  in  fact,  in  almost  any  condition  giving  rise  to  a  concentrated 
urine. 

Sodium  Urate. — Sodium  urate  (Fig.  10)  may  occur  either  in  an 
amorphous  or  a  crystalline  state.  When  crystalline,  it  appears 
in  sheaves  or  clusters  of  colorless  needles,  resembling  calcium 
phosphate  crystals,  from  which  they  may  be  differentiated  as 
already  described. 

Ammonium  Urate. — This  compound  (Fig.  11)  occurs  in  mark- 
edly alkaline  urines  as  yellow  spherical  groups  of  very  fine 
needles,  or  in  a  u  thorn  apple  "  form,  which  appears  to  be 
balls  with  spicules  of  various  sizes  attached. 

Cystine  crystallizes  as  thin  colorless  hexagonal  plates ;  tyrosine, 
as  long  very  thin  needles  grouped  in  sheaves;  and  leucine,  as 
spherules  resembling  fat  droplets,  from  which  they  can  be  distin- 
guished by  their  insolubility  in  ether,  and  by  the  detection  of 
concentric  striations  and  radiating  lines. 

URINARY    CALCULI. 

In  many  instances,  unorganized  urinary  sediments  appear  in 
the  urine  only  after  voiding.  When  such  deposits  regularly  form 
within  the  urinary  tract,  they  are  liable  to  gather  about  some 
material  which  acts  as  a  nucleus  (mucus,  epithelial  cells,  bac- 
teria, etc.)  and  form  calculi.  Almost  any  of  the  unorganized  sedi- 
ment constituents  may  enter  into  the  formation  of  such  calculi,  but 
calcium  oxalate,  calcium  phosphate  and  uric  acid  are  the  most 
common.  It  has  long  been  supposed  that  urinary  calculi  were 
largely  composed  of  uric  acid.  Recent  analyses,  however,  do  not 
appear  to  entirely  support  this  idea.  Kahn  and  Rosenbloom1  re- 
port analyses  of  sixteen  renal  calculi,  which  were  composed  mostly 
of  calcium  oxalate,  and  two  cystic  calculi  which  were  almost  pure 
uric  acid.  From  the  point  of  view  of  therapy,  it  is  desirable  to 
determine  whether  we  are  dealing,  on  the  one  hand,  with  uric 
acid,  or,  on  the  other,  with  calcium  oxalate  and  calcium  phosphate 
calculi,  since  diametrically  opposite  treatment  should  be  in- 
stituted. The  two  types  of  calculi  may  be  differentiated  by  their 
action  on  ignition  and  treatment  with  hydrochloric  acid.     Uric 


1.  Kahn  and  Rosenbloom:  Jour.  Amer.  Med.  Assoc,  1912,  LIX,  p.  2252; 
also  Rowlands:   Biochem.  Jour.,  1908,   III,  p.  346. 


102  PATHOLOGICAL  CHEMISTRY 

acid  is -insoluble  in  the  acid  and  leaves  but  little  residue  on  igni- 
tion; While  the  oxalate  and  phosphate  of  lime  are  soluble  in  this 
acid  and  are  not  appreciably  affected  by  ignition.  Since  uric 
acid  calculi  are  soluble  in  alkalies  and  insoluble  in  acids,  whereas 
the  reverse  relation  holds  for  the  lime  concretions,  it  is  obvious 
that  alkaline  therapy  cannot  bring  about  solution  of  calculi 
made  up  for  the  most  part  of  calcium  oxalate  and  calcium  phos- 
phate, such  treatment  being  indicated  only  for  uric  acid  calculi. 
For  consideration  of  other  forms  of  calculi,  and  for  details  on  the 
analysis  of  calculi,   reference  may  be  made   to   other  works.1 

LABORATORY    PROCEDURES. 

The  urinary  sediment  should  be  examined  as  soon  as  possible 
after  voiding.  When  a  centrifuge  is  available  this  may  be  done  at 
once,  otherwise,  it  is  advisable  to  allow  the  urine  to  remain  for 
some  hours  in  a  conical  sedimenting  cylinder.  Where  this  is 
done,  it  is  necessary  to  take  precautions  to  preserve  the  specimen, 
e.g.,  by  refrigeration,  or  with  toluene,  since  casts  and  leucocytes 
are  liable  to  undergo  degenerative  changes.  The  supernatant 
liquid  in  the  centrifuge  tube  may  be  readily  removed  by  simply 
inverting,  most  of  the  sediment  remaining  in  the  tip.  The  latter 
is  then  agitated,  the  one  or  two  drops  of  sediment  transferred 
to  and  spread  out  on  a  broad  glass  slide,  and  examined  under  the 
microscope.  In  searching  for  casts,  the  low  power  objective  and 
high  power  eye-piece  should  be  employed  and  the  light  reduced 
as  far  as  possible  with  the  iris  diaphragm.  This  is  especially  im- 
portant in  the  search  for  hyaline  casts,  as  these  appear  as  mere 
shadows.  For  a  careful  observation  of  erythrocytes,  bacteria, 
etc.,  the  high  power  objective  is  preferable;  and  if  more  than  a 
mere  inspection  is  to  be  made,  it  will  be  found  convenient  to 
employ  a  cover  glass.  When  the  casts  are  impregnated  with  red 
cells,  leucocytes,  epithelial  cells,  etc.,  they  become  more  plainly 
visible.  Occasionally  deposits  of  phosphates  may  be  mistaken 
macroscopically  for  pus,  but  the  former  will  disappear  on  the 
addition  of  acetic  acid,  whereas  the  pus  sediment  is,  if  anything, 
accentuated  under  these  conditions.  Where  similarity  of  cry- 
stalline forms  causes  confusion,  advantage  may  be  taken  of  solu- 
bility reactions  as  already  described. 

1.  Purdy:  Practical  Uranalysis  and  Urinary  Diagnosis,  Philadelphia, 
1901,  p.  241;  Wood:  Chemical  and  Microscopical  Diagnosis,  New  York, 
1911,  p.  608. 


CHAPTER    IX.    . 
The  Chemistry  and  Physiology  of  Milk. 

From  the  chemical  point  of  view,  the  differences  observed  in 
the  milk  of  various  species,  are  primarily  quantitative,  although 
these  may  assume  considerable  physiological  importance.  For 
this  reason,  attention  will  be  devoted  first  to  the  properties  and 
constituents  of  milk,  irrespective  of  its  origin,  and  then  to  a 
consideration  of  its  adaptation  to  the  nutrition  of  various  animals. 

Milk  is  essentially  an  emulsion  of  finely  divided  fat  in  a 
solution  of  protein,  sugar,  mineral  matter,  organic  extractives 
and  enzymes.  It  is  the  emulsified  fat,  together  with  a  fine 
suspension  of  a  casein-lime  combination,  which  gives  to  milk  its 
characteristic  yellowish  white  non-transparent  appearance.  The 
specific  gravity  of  milk  varies  between  1.028  and  1.034.  The 
reaction  of  perfectly  fresh  milk  is  amphoteric  toward  litmus. 
Attempts  have  been  made  to  determine  the  exact  degree  of 
acidity  or  alkalinity  by  titration  methods,  but  it  is  probable  that 
milk,  like  body  fluids  in  general,  is  essentially  neutral,  that  is, 
there  is  no  noteworthy  preponderance  of  the  hydrogen  ion  or 
of  the  hydroxyl  ion.  On  standing,  there  is  a  progressive  increase 
in  the  acidity,  owing  to  the  action  of  bacteria  upon  the  milk  sugar 
by  which  lactic  acid  is  formed.  Fresh  milk  does  not  coagulate 
on  boiling,  but  forms  a  thin  pellicle  consisting  of  casein  and  lime 
salts.  As  the  bacterial  formation  of  lactic  acid  progresses,  the 
ease  with  which  coagulation  occurs  increases,  and  finally,  when 
the  concentration  of  lactic  acid  is  sufficiently  great,  coagulation 
takes  place  spontaneously  at  room  temperature. 

Qualitative  Composition. — Fat  exists  in  milk  in  the  form  of  very 
small  globules.  Although  the  composition  of  milk  fat  (butter) 
varies  considerably  under  different  circumstances,  it  is  composed 
chiefly  of  olein  and  palmitin,  together  with  small  amounts  of 
the  glycerides  of  butyric  acid  and  of  several  other  fatty  acids. 
In  addition  to  these  there  are  present  small  quantities  of  lecithin, 
cholesterol  and  a  yellow  coloring  matter.  Attempts  have  been 
made  to  alter  the  proportion  of  olein,  palmitin  and  stearin  of 
ordinary  beef  fat  in  such  a  manner  that  when  mixed  with  lard 

103 


104  PATHOLOGICAL  CHEMISTRY 

and  small  quantities  of  milk  and  pure  butter  a  product  very  similar 
to  butter  is  obtained.  This  is  known  commercially  as  oleo- 
margarine. The  properties  of  the  latter  do  in  fact  closely 
resemble  those  of  butter,  although  it  is  very  much  lower  in  the 
glycerides  of  the  volatile  fatty  acids,  e.g.,  butyrin.  Nevertheless, 
disregarding  the  question  of  palatability,  there  appears  to  be  no 
reason  why  such  a  preparation  should  not  have  a  nutritive  value 
equivalent  to  butter,  and,  indeed,  for  the  diabetic  the  low  con- 
centration of  butyric  acid  would  make  it  preferable.  (See 
Chapter  VI.) 

Protein  is  present  in  milk  as  casein  and  lactalbumin.  The 
latter  differs  in  no  important  detail  from  ordinary  albumins,  but 
the  casein  presents  certain  unique  features.  It  belongs  to  the 
class  of  phosphoproteins,  which  are  characterized  by  their  con- 
tent of  phosphorus.  Casein  may  be  precipitated  from  milk  by 
rendering  the  latter  slightly  acid  with  acetic  acid,  an  excess  of 
the  acid  subsequently  dissolving  the  precipitate.  Lactic  acid 
resulting  from  bacterial  action  on  lactose  may  likewise  precipitate 
the  casein.  Casein  thus  prepared  and  dried  is  not  appreciably 
soluble  in  pure  water,  but  forms  soluble  combinations  with 
alkalies  and  alkali  earths,  thus  demonstrating  its  acid  nature. 
Such  a  combination  with  calcium,  mixed  with  calcium  phosphate, 
presents  a  whitish  opalescent  appearance;  and  it  is  not  improb- 
able that  the  casein  exists  in  milk  in  this  form  and  so  contributes 
to  the  opaque  character  of  this  fluid.  The  most  characteristic 
property  of  casein  is  its  coagulation  under  the  influence  of 
the  enzyme,  rennin,  for  which  the  presence  of  lime  salts  is 
essential.  The  enzymatic  clotting  of  casein  takes  place  in 
neutral  medium  and  should  be  distinguished  from  acid  coagula- 
tion. It  is  probable  that  the  action  of  rennin  and  lime  salts  is 
distinct,  the  enzyme  acting  upon  the  casein  to  form  the  slightly 
hydrolyzed  product  M  paracasein,"  which  then  forms  an  insoluble 
compound  with  the  lime  and  is  precipitated.  This  can  be  demon- 
strated by  permitting  rennin  to  act  upon  lime-free  casein  solu 
tions.  No  precipitate  is  formed.  If  the  mixture  is  boiled, 
thus  killing  the  enzyme,  and  subsequently  calcium  salts  added, 
the  usual  clotting  is  observed.  .  A  number  of  commercial  prep 
arations  of  milk  proteins  have  appeared,  e.g.,  u  nutrose  "— 
sodium  salt  of  casein;  "  eucasein  " — the  ammonium  salt  of 
casein;  "  plasmon  " — a  mixture  of  casein  and  sodium  carbonate 
"  lactalbumin  "  and  M  albuminlac." 


COMPOSITION   OF   MILK 


105 


The  sugar  of  milk  is  lactose.  Its  molecule  consists  of  one 
molecule  each  of  glucose  and  galactose.  Many  of  the  properties 
of  lactose  have  been  described  in  Chapter  V.  Under  the  influence 
of  certain  bacteria,  lactose  is  decomposed  into  lactic  acid,  to  which 
milk  owes  its  increasing  acidity  on  standing.  Owing  to  the  action 
of  certain  yeasts  and  bacteria,  a  combination  of  alcoholic  and 
lactic  acid  fermentation1  of  lactose  is  induced  with  the  produc- 
tion of  M  kumyss  "  and  "  kephir,"  the  former  from  mare's  milk 
and  the  latter  from  cow's  milk. 

The  mineral  matter  of  milk  consists  of  the  phosphates  and 
chlorides  of  calcium,  magnesium,  sodium,  potassium  and  iron, 
although  portions  of  some  of  these  metals  are  probably  in  organic 
combination.  Considerable  interest  is  attached  to  the  iron  as  it 
exists  in  the  milk  in  extremely  small  amounts  (0 .  0003-0 .  004  per 
cent.) .  This  deficiency,  however,  is  compensated  by  an  abundant 
supply  of  iron  in  the  infant's  liver. 

The  organic  extractives  of  milk  include  traces  of  urea,  creatine, 
creatinine,  orotic  acid  and  citric  acid. 

Quantitative  Relations. — For  the  quantitative  composition  of 
cow's  milk,  which  has  been  the  subject  of  more  numerous  analyses 
than  any  other,  the  following  may  be  taken  as  average  figures: 
water,  87  per  cent.;  protein,  3.5  per  cent.;  fat,  3.7  per  cent.; 
sugar,  4.9  per  cent.,  and  ash,  0.7  per  cent.  It  is  interesting 
to  note  that  the  composition  of  the  milk  of  various  animals 
shows  certain  definite  relations  to  the  rate  of  growth  of  the  young 
of  the  species.    This  is  illustrated  in  the  following  table: 


Species 

Number  of  days  required 

to  double  weight  at 

birth 

Percentage  composition 
(partial)  . 

of  milk 

Protein 

Ash 

Sugar 

Human 

180 

1.6 

0.2 

7.0 

Horse 

60 

2.0 

0.4 

6.7 

Cow 

47 

3.5 

0.7 

4.9 

Goat 

22 

3.7 

0.8 

4.4 

Sheep 

15 

4.9 

0.8 

4.0 

Swine 

14 

5.2 

0.8 

4.0 

Dog 

9 

7.4 

1.3 

3.2 

Rabbit 

6 

14.4 

2.5 



1.    Some  protein  decomposition  also  probably  takes  place. 


106  PATHOLOGICAL  CHEMISTRY 

It  will  be  observed  that  for  the  infant,  who  doubles  its  initial 
weight  in  180  days,  the  milk  is  relatively  low  in  protein  and 
mineral  constituents,  whereas  for  the  rabbit,  which  grows  very 
rapidly,  doubling  its  weight  in  but  six  days,  the  protein  and 
ash  of  the  milk  are  very  high.  The  animals  intermediate  in 
the  list  show  corresponding  variations.  It  is  obvious  that  the 
more  rapidly  an  animal  grows,  involving  the  formation  of  muscle 
and  skeletal  tissue,  the  greater  will  be  its  requirement  of  protein 
and  mineral  matter. 

Of  practical  interest  is  the  difference  in  composition  between 
human  and  cow's  milk.  Although  both  types  show  marked 
variations  in  composition,  it  may  be  said  that,  in  general,  cow's 
milk  contains  twice  as  much  protein,  three  times  as  much  mineral 
matter,  and  considerably  less  sugar,  than  does  woman's  milk. 
Another  important  difference  is  the  form  of  protein.  Whereas 
cow's  milk  contains  about  15  per  cent,  of  its  protein  in  the 
form  of  lactalbumin,  the  lactalbumin  constitutes  nearly  40  per 
cent,  of  the  protein  in  human  milk.  Further,  the  fat  of  mother's 
milk  differs  from  that  of  cow's  milk  in  that  it  contains  more 
olein.  These  differences  become  important  considerations  when 
it  is  desired  to  substitute  cow's  milk  for  mother's  milk,  certain 
modifications  of  the  former  being  essential.  Before  further 
reference  is  made  to  methods  of  milk  modification,  attention 
will  be  called  to  a  few  of  the  fundamental  metabolic  require- 
ments of  the  infant. 

Heat  production  is  proportional  to  the  surface  of  the  body 
rather  than  to  body  weight.  Hence,  since  infants  have  greater 
surfaces  relative  to  their  body  weights  than  do  adults,  it  follows 
that  they  have  a  greater  calorific  requirement  per  unit  weight. 
Thus  a  child  under  one  year  should  receive  at  least  100  calories 
per  kilo  body  weight,  the  calorific  intake  being  gradually  reduced 
until  adult  life,  when  35  to  60  calories  per  kilo  should  prove  ade- 
quate. It  is  important  that  the  infant  be  supplied  with  a  suffi- 
cient number  of  calories,  as  it  is  only  under  this  condition  that 
the  maximum  degree  of  nitrogen  retention  and  the  proper  rate 
of  growth  can  be  attained.  In  human  milk  the  protein  supplies 
about  9  per  cent,  of  the  calories,  and  in  general  this  relation  may 
advantageously  be  preserved  in  the  dietary  throughout  life. 
For  example,  for  a  man  at  moderately  active  work  a  diet  con- 
taining 75  grams  of  protein  and  furnishing  3000  calories  would 


COMPOSITION   OF   MILK  107 

be  ample.  Here  the  protein  supplies  10  per  cent,  of  the  total 
calories.  Because  of  the  relatively  low  concentration  of  sugar 
and  relatively  high  concentration  of  protein  in  cow's  milk,  it  is 
difficult  to  supply  the  infant  with  the  requisite  number  of  calories 
without  at  the  same  time  giving  too  great  a  quantity  of  protein. 
Cow's  milk  contains  about  20  per  cent,  of  its  calories  in  the  form 
of  protein,  an  amount  larger  than  the  infant  can  properly  digest. 
Moreover,  according  to  Howland,1  when  the  proportion  of 
protein  reaches  this  figure,  the  specific  dynamic  action  becomes 
very  marked.  Howland  states  that  protein  should  normally 
furnish  not  less  than  8  or  more  than  10  per  cent,  of  the  total 
energy  of  the  food. 

The  "  top  milk  "  method  is  at  present  the  most  commonly 
employed  procedure  for  modifying  cow's  milk,  and  is  briefly  as 
follows :  the  milk  in  a  quart  bottle  is  allowed  to  stand  and  accum- 
ulate its  layer  of  cream  in  the  upper  part  of  the  bottle.  The  com- 
position of  this  layer  is  essentiallv  altered  only  with  respect  to 
fat,  which  may  reach  25  per  cent.,  whereas  the  concentrations 
of  protein,  sugar,  and  salts  remain  practically  unchanged.  By 
utilizing  the  upper  part  of  the  contents  of  the  bottle  to  varying 
depths  below  the  layer  of  cream  it  is  possible  to  obtain  milk  of 
any  desired  fat  concentration.  For  the  relation  between  depth 
and  resulting  fat  concentration,  we  must  refer  to  works  on 
pediatrics.2  By  properly  diluting  milk  of  such  known  fat 
concentrations,  one  may  obtain  any  desired  concentration  of 
protein  and  fat,  the  deficiency  in  sugar  being  made  up  by  appro- 
priate additions  of  this  material.  In  many  of  the  large  cities 
there  are  laboratories,  which  undertake  to  supply  milk  of 
any  composition  according  to  the  physician's  prescription. 
However,  the  "  top  milk  "  method  of  modifying  milk  must  still 
be  generally  employed,  as  such  laboratories  are  not  yet  suffi- 
ciently widely  distributed.  In  addition  to  the  general  laws  of 
infant  nutrition  as  above  enunciated,  there  are  numerous  indi- 
vidual requirements  of  the  infant  which  can  be  learned  only  by  a 
careful  study  of  each  case.  At  times,  for  example,  it  may  not  be 
sufficient  to  simply  make  the  percentage  composition  of  cow's 
milk  resemble  mother's  milk.     Even  when  thus  modified,  the 


1.  Howland:   Amer.  Jour.  Dis.  Child.,  1911,  II,  p.  49. 

2.  Cf.     H.  D.  Chapin   and   G.    R.    Pisek:    "  Diseases   of  Infants  and 
Children,"  1911,  New  York. 


108  PATHOLOGICAL  CHEMISTRY 

proportion  of  protein,  or  sugar,  or  fat  may  be  too  great  or  too 
small;  or  a  sugar  other  than  lactose  (e.g.,  cane  sugar  or  maltose) 
may  be  better  tolerated.  There  appears  to  be  a  tendency  at 
present  toward  high  protein  feeding.1  For  further  discussion 
on  infant  feeding  we  must  refer  to  other  works.2 

Aside  from  the  quantitative  variations  between  human  and 
cow's  milk  there  are  certain  qualitative  differences.  The  casein 
of  woman's  milk  forms  a  much  less  dense  coagulum  under  the 
influence  of  rennin  than  does  cow's  milk,  a  condition  which 
accounts  for  the  relative  ease  with  which  mother's  milk  is 
digested  by  the  infant. 

Secretion  of  Milk. — The  initiation  of  milk  secretion  appears  to 
be  independent  of  the  nervous  system,  although  normally  the 
latter  undoubtedly  exerts  a  regulatory  influence.  This  view  is 
the  result  of  experiments  on  animals  in  which  milk  secretion  was 
not  prevented  when  all  nerves  to  the  mammary  glands  were  cut. 
From  the  work  of  Starling  and  Lane-Claypon  it  would  appear 
that  the  development  of  secreting  cells  is  stimulated  by  some 
hormone  influence.  They  found  that  injections  of  fetal  extracts 
into  virgin  rabbits  induce  growth  in  the  mammary  gland,  from 
which  they  concluded  that  normally  the  fetus  elaborates  a 
hormone  which  stimulates  the  growth  of  the  mammary  gland, 
but  inhibits  secretion.  After  birth,  when  the  source  of  the  hor- 
mone is  removed,  lactation  starts,  but  is  again  arrested  should 
pregnancy  intervene  before  the  end  of  lactation. 

During  the  last  few  days  of  pregnancy  and  the  first  three  or 
four  days  after  parturition,  the  mammary  glands  secrete  a  fluid 
(colostrum),  which  differs  from  true  milk.  It  is  of  a  deeper 
yellow  color  than  milk,  and  has  a  higher  specific  gravity  which 
may  be  as  great  as  1 .060.  It  is  lower  in  fat  and  sugar  than  milk, 
but  is  considerably  richer  in  protein,  which  accounts  for  its 

1.  At  present  pediatricians  are  not  inclined  to  regard  the  high 
proportion  of  casein  in  cow's  milk  as  detrimental  to  the  digestive  appara- 
tus of  the  healthy  infant,  although  prolonged  high  protein  feeding  would 
appear  to  maintain  the  intensity  of  metabolism  at  an  unnecessarily  high 
level.  In  the  healthy  infant  there  appears  likewise  lo  be  no  essential 
difference  in  the  tolerance  for  the  various  sugars.  (See  Howland:  Harvey 
lecture,  1913.) 

2.  Cf.  H.  D.  Chapin  and  G.  R.  Pisek:  loc  cit.\  also  Langstein  and 
Meyer:  "  Sauglingsernahrung  und  Sauglingsstoffwechsel,"  Wiesbaden, 
1910. 


COMPOSITION   OF   MILK  109 

ability  to  coagulate  on  heating.  In  addition,  colostrum  contains 
an  abundance  of  nucleated  granular  cells — the  colostrum  cor- 
puscles. A  few  days  after  delivery  the  colostrum  takes  on  the 
appearance  and  composition  of  true  milk,  although  it  may  be- 
several  weeks  before  a  constant  composition  is  attained.  During 
the  first  few  weeks  of  lactation,  the  volume  of  the  secretion  is 
commonly  as  great  as  400  cc,  furnishing  about  250  calories; 
but  as  the  child  grows,  the  secretion  increases  and  may  furnish  as 
much  as  750  calories. 

Milk  is  the  result  of  a  specific  secretory  activity  of  the  cellular 
elements  of  the  mammary  gland,  filtration  and  diffusion  being  of 
secondary  importance  in  this  connection.  This  is  indicated  by 
the  fact  that  milk  contains  lactose,  whereas  blood  contains  glu- 
cose. Moreover,  casein  is  not  found  in  blood,  and  lactal- 
bumin  is  not  identical  with  seralbumin.  Again,  according  to 
Bunge,  the  mineral  constituents  of  milk  are  present  in  different 
proportion  than  those  in  serum.  The  fat  is  no  doubt  likewise 
elaborated  in  the  cells  of  the  mammary  gland,  but  may  also  be 
drawn  from  other  fat  depots  in  the  body.  As  to  the  direct 
passage  of  food  fat  into  the  secretion  there  is  considerable 
difference  of  opinion.  Certainly  some  ingested  substances  can 
find  their  way  into  the  milk.  This  is  true  of  substances  of  the 
food  which  impart  certain  unusual  flavors  to  the  milk,  and  of 
drugs,  e.g.,  morphine,  quinine,  iodides,  arsenic,  lead,  mercury, 
iron,  etc. 

With  regard  to  the  influence  of  food  upon  the  composition  of 
milk,  we  may  say  that  an  insufficient  diet  decreases  both  the 
quantity  of  milk  and  the  solids,  while  both  are  increased  by 
abundant  food.  Lactose  appears  to  vary  less  under  the  influence 
of  food  than  protein  and  fat.  On  the  whole,  the  diet  appears  to 
be  of  secondary  importance  in  this  connection;  the  secretion  of 
milk  is  an  individual  variable,  and  subject  to  nervous  influences. 

Sterilization  and  Pasteurization  of  Milk. — Milk  properly 
sterilized  in  an  autoclave  is  free  from  all  living  organisms,  but 
simply  bringing  to  a  boil,  as  "  sterilization  "  is  ordinarily  per- 
formed in  the  household,  does  not  suffice  for  the  destruction  of 
spores,  although  the  lactic  acid  producing  organisms  are  probably 
destroyed.  Thus  milk  after  being  subjected  to  the  usual  home 
sterilization  is  not  so  likely  to  "  sour,"  but  may,  nevertheless, 
harbor    numerous    non-acid    producing    organisms.      Heating 


110  PATHOLOGICAL  CHEMISTRY 

induces  certain  changes  in  the  milk.  The  casein  becomes  less 
digestible,  and  undergoes  partial  decomposition  associated  with 
the  liberation  of  a  volatile  sulphide.  Any  enzymes  present 
would,  of  course,  be  destroyed.  Exactly  to  what  extent  these 
changes  are  deleterious  is  not  certain,  but  it  is  significant  to  note 
that  a  large  proportion  of  cases  of  scurvy  have  been  attributed  to 
the  use  of  sterilized  milk.  Pasteurization — heating  to  68°  C. 
for  thirty  minutes — kills  most  vegetative  organisms  but 
•not  spores.  It  has  the  advantage  that  there  is  less  alteration  in 
the  milk  constituents,  but  the  keeping  quality  of  such  milk  is 
not  so  great  and  it  must  therefore  be  preserved  at  a  low  tem- 
perature before  consumption. 

LABORATORY   PROCEDURES. 

The  chemical  examination  of  either  human  or  cow's  milk 
is  necessarily  a  quantitative  one,  in  which  determinations  of 
the  various  constituents,  protein  (casein  and  lactalbumin) , 
fat,  lactose  and  ash,  are  made  to  ascertain  if  they  exist  in  the 
normal  proportions.  As  has  been  previously  pointed  out,  human 
milk  normally  contains  87  to  88  per  cent,  of  water,  3  to  4  per  cent, 
of  fat,  5  to  8  (6  average)  per  cent,  of  lactose,  1  to  2  per  cent,  of  pro- 
tein and  0.2  to  0.4  per  cent.  ash.  It  has  a  pale  blue  color,  a 
specific  gravity  between  1 .  028  and  1 .  032  and  is  amphoteric 
in  reaction.  The  average  composition  of  cow's  milk  from 
a  large  series  of  American  analyses  is  87.4  per  cent,  of 
water,  3.5  per  cent,  of  fat,  4.5  per  cent  of  lactose,  3.9 
per  cent,  of  proteins  and  0.7  percent,  of  ash,  with  a  specific 
gravity  of  1.031.  Human  milk  is  thus  seen  to  contain  more 
lactose  and  much  less  protein  and  inorganic  salts.  When  an 
infant  does  not  appear  to  be  following  the  usual  growth  curve 
or  where  digestive  disorders  are  manifest,  an  analysis  of  the 
milk  of  the  mother  may  indicate  the  cause  of  the  abnormality. 
One  would  thus  be  in  a  position  to  decide  whether  cow's  milk 
should  be  substituted  and  to  what  extent  the  latter  should 
be  modified. 

Holt  describes  a  small  milk-testing  apparatus  consisting  of 
two  cylinders,  one  for  ascertaining  the  specific  gravity,  the  other 
for  roughly  estimating  the  fat  by  the  amount  of  cream  which  rises 
in  a  given  length  of  time.  This  may  in  certain  instances  be  of 
value  from  a  clinical  standpoint  on  account  of  its  simplicity. 


MILK  ANALYSIS  111 

Even  such  simple  determinations  as  the  specific  gravity  often 
yield  very  valuable  results,  as  in  the  detection  of  adulteration 
of  cow's  milk.  It  is  likewise  of  importance  at  times  to  test  for 
certain  of  the  preservatives  which  are  sometimes  added  to  milk, 
such  as  formaldehyde,  hydrogen  peroxide  and  boric  acid. 

1 .  Reaction. — This  may  be  determined  qualitatively  with 
red  and  blue  litmus  paper.  Normally,  it  is  slightly  alkaline  in 
its  reaction  to  red  litmus  paper. 

The  amount  of  acidity  may  be  titrated  with  N/10  NaOH, 
using  phenolphthalein  as  an  indicator.  Ten  cc.  of  milk  are 
measured  into  an  evaporating  dish  and  diluted  with  50  cc.  of 
water  and  2  to  3  drops  of  1  per  cent,  solution  of  phenolphthalein 
added.  The  acidity. may  be  expressed  in  degrees  by  considering 
each  cc.  of  N/10  NaOH  required  to  neutralize  *  100  cc.  of 
milk  as  one  degree.  An  acidity  of  15  to  25  degrees  may  be  ob- 
served within  six  hours  after  milking,  and  forty-eight  hours  after 
the  acidity  may  be  as  high  as  100  degrees.  In  buttermilk  the 
acidity  is  generally  between  100  and  125. 

2 .  Specific  Gravity. — The  specific  gravity  of  milk  may  be 
determined  either  with  a  special  lactometer,  or  with  an  ordinary 
urinometer.  The  temperature  of  the  milk  should  be  60  degrees 
F.  A  rough  correction  can  be  made  by  adding  0.0001  to  the 
reading  for  each  degree  above  60  degrees  F.,  and  subtracting 
the  same  amount  for  each  degree  below. 

3 .  Total  Solids. — The  total  solids  of  the  milk  may  be  roughly 
calculated  from  the  lactometer  readings  by  the  following  formula 
suggested  by  Babcock.  L  =  last  two  figures  of  the  specific 
gravity  corrected  for  temperature.  F  =  the  percentage  of  fat 
found  in  the  milk. 

Total  solids  =  L/4  +  0.2   F  +  F. 

4.  Fat. — The  fat  content  in  human  milk  may  be  determined 
with  a  fair  degree  of  accuracy  with  the  small  Babcock  tube  which 
fits  the  conical  cup  of  the  ordinary  medical  centrifuge.  It 
requires  5  cc.  of  milk,  an  equal  volume  of  sulphuric  acid,  specific 
gravity  1 .83,  and  enough  of  a  mixture  of  equal  parts  of  concen- 
trated hydrochloric  acid  and  amyl  alcohol  to  fill  the  tube. 
Five  cc.  of  the  thoroughly  mixed  milk  are  pipetted  into  the  tube, 
the  above  solutions  added,  the  tube  centrifuged  for  five  minutes 
and  the  percentage  of  fat  read  off  directly  on  the  tube.    Where 


112  PATHOLOGICAL  CHEMISTRY 

sufficient  mother's  milk  is  available,  or  in  the  case  of  cow's 
milk,  the  large  Babcock  tube  for  17  .6  cc.  of  milk  should  be  em- 
ployed as  the  results  are  more  satisfactory.  This  amount  of 
milk  is  placed  in  the  tube  with  the  special  pipette,  17.5  cc. 
sulphuric  acid,  specific  gravity  1 .82  to  1 .83,  poured  gently  down 
the  side  of  the  tube  and  the  tube  mixed  by  a  combination  of  a 
rotary  and  shaking  motion.  The  tube  is  at  once  centrifugalized 
for  five  minutes,  then  boiling  water  added  to  the  tube  to  bring 
the  fat  up  into  the  graduated  portion  of  the  neck.  The  tube  is 
again  centrifugalized  for  one  minute  and  the  content  of  fat  read 
off  on  the  tube. 

5 .  Protein. — 

a.  Kjeldahl  Method. — The  total  protein  may  be  accurately 
estimated  by  determining  the  nitrogen  in  carefully  measured 
samples  of  milk  (5  cc.)  by  the  Kjeldahl  method  as  previously 
described1  under  Urine.  Human  milk  contains  about  9  per 
cent,  non-protein  nitrogen  and  cow's  milk  about  6  per  cent. 
This  is  deducted  from  the  nitrogen  obtained,  and  the  result 
multiplied  by  6.34  to  ascertain  the  amount  of  protein. 

b.  Precipitation  with  Phosphotungstic  Acid. — Five  cc.  of  milk 
(or  if  necessary  1  cc.)  are  diluted  with  9  parts  of  water  and 
thoroughly  mixed.  This  is  poured  into  an  ordinary  Esbach 
albuminometer  to  the  mark  U  and  a  phosphotungstic  acid 
solution  2  added  to  R.  The  tube  is  then  stoppered  and  inverted 
several  times  to  insure  a  thorough  mixing.  It  is  now  set  aside 
for  24  hours  at  room  temperature  and  the  percentage  of  protein 
read  off  directly.  This  method  gives  k  fair  degree  of  accuracy, 
especially  in  woman's  milk  where  the  amount  of  casein  is  low.3 

6.  Lactose. — To  10  cc.  of  milk  are  added  10  cc.  of  the  phos- 
photungstic acid  solution  employed  above  and  20  cc.  of  water. 
(If  material  available  is  insufficient,  half  quantities  of  the  milk 
and  other  solutions  may  be  taken.)  After  thoroughly  mixing, 
this  material  is  filtered  through  a  dry  filter.  The  filtrate  comes 
through  perfectly  clear  and  the  lactose  content  of  this  filtrate 
may  be  determined  either  polariscopically  (specific  rotation  of 
lactose  +  53.0)  or  by  titration  with  Benedict's  solution.4     The 

1.  See  Chapter  III,  p.  44. 

2 .  The  phosphotungstic  acid  solution  is  prepared  by  dissolving  70  grams 
of  phosphotungstic  acid  in  distilled  water  containing  20  cc.  of  concentrated 
hydrochloric  acid  and  making  up  to  one  liter. 

3.  Cf.     Boggs:    Johns  Hopkins  Hospital  BuL,  1906,  XVII,  p.  342. 

4.  See  Chapter  V,  p.  76. 


MILK  ANALYSIS  113 

titration  is  carried  out  the  same  as  for  glucose  in  urine.  In  this 
case,  however,  we  have  found  that  0.067  gm.  lactose  is  required 
to  reduce  the  25  cc.  of  Benedict's  solution.1  In  making  the 
calculation,  the  polariscopic  figure  is  multiplied  by  four,  or  the 
titration  figure  divided  by  four,  to  allow  for  the  dilution. 
7 .  Detection  of  Preservatives. — 

a.  Leach's  Hydrochloric  Acid  Test  for  Formaldehyde. — Add 
10  cc.  of  the  acid  reagent2  to  an  equal  volume  of  the  milk  in  a 
porcelain  casserole,  and  heat  slowly  over  the  free  flame  nearly 
to  boiling,  holding  the  casserole  by  the  handle,  and  giving  it  a 
rotary  motion  while  heating  to  break  up  the  curd.  The  presence 
of  formaldehyde  is  indicated  by  a  violet  coloration,  varying  in 
depth  with  the  amount  present.  In  the  absence  of  formaldehyde, 
the  solution  slowly  turns  brown.  By  this  test  1  part  of  formalde- 
hyde in  250,000  parts  of  milk  is  readily  detected  before  the  milk 
sours.    After  souring  the  delicacy  is  reduced  to  1-50,000. 

b.  Hydrogen  Peroxide. — Upon  adding  2  to  3  drops  of  a  2  per 
cent,  aqueous  solution  of  />-phenylenediamine  hydrochloride 
to  about  10  cc.  of  milk,  a  blue  color  will  immediately  be  pro- 
duced in  the  presence  of  hydrogen  peroxide  upon  shaking  or 
allowing  the  mixture  to  stand  for  a  few  minutes.  A  delicacy  of 
1  part  in  40,000  is  claimed  for  this  method. 

c.  Boric  Acid. — The  turmeric-paper  test  may  be  applied 
either  to  the  ash  or  directly  to  the  milk.  In  the  latter  case  10 
cc.  of  milk  are  thoroughly  mixed  with  6  drops  of  concentrated 
hydrochloric  acid,  the  turmeric  paper  moistened  with  the  mixture 
and  then  dried.  The  presence  of  boric  acid  is  indicated  by  the 
production  of  a  deep  red  color,  which  is  changed  to  green  or  blue 
upon  treatment  with  dilute  alkali.  The  test  when  properly  ap- 
plied has  a  delicacy  of  1  part  in  8000. 

1.  Myers:    Munch,  med.  Wochenschr.,  1912,  LIX,  p.  1494. 

2.  Commercial  hydrochloric  acid  (sp.  gr.  1.2)  containing  2  cc.  of 
10  per  cent,  ferric  chloride  per  liter  is  used  as  reagent. 


CHAPTER  X. 
Blood  and  Other  Body  Fluids. 

The  present  chapter  will  be  confined  to  a  consideration  of  the 
chemistry  of  body  fluids,  although  space  will  allow  only  a  brief 
discussion  of  even  this  aspect  of  the  subject.  For  this  reason 
attention  will  be  directed  primarily  to  certain  clinical  possibilities 
opened  up  by  several  recent  American  investigations. 

Blood. — From  6  to  8  per  cent,  of  the  weight  of  the  body  is 
made  up  by  blood.  In  a  certain  sense  it  may  be  regarded  as  a 
fluid  tissue,  consisting  of  a  transparent  amber  colored  liquid 
(the  blood  plasma) ,  in  which  are  suspended  an  enormous  number 
of  formed  elements — the  erythrocytes,  leucocytes  and  blood 
platelets.  The  specific  gravity  of  blood  varies  between  1.045 
and  1.075;  for  adult  men  the  average  is  1.058,  and  a  little 
less  for  women.  Blood  is  alkaline  toward  litmus,  but  from  the 
physico-chemical  point  of  view  it  is  essentially  neutral. 

The  chemical  composition  of  blood  plasma  may  be  briefly 
outlined  as  follows : 


Plasma 


Protein 
(fibrinogen) 


Serum 


Proteins 


Organic 
extractives 


Salts 


Enzymes 


Internal 
secretions,  etc. 


seralbumin, 
serglobulin. 

glucose,  fats,  lipoids, 
urea,  uric  acid,  etc. 

chlorides,  carbonates,  sul- 
phates, and  phosphates  of 
ammonium,  potassium,  cal- 
cium, magnesium  and  iron. 

thrombin,  oxidase,  catalase, 
amylase,  lipase,  etc.,  antien- 
zymes. 


The  most  striking  property  of 'blood,  or  of  blood  plasma,  is  its 
ability  to  clot.     This  process  involves  the  conversion  of  the 


114 


COMPOSITION   OF  BLOOD  115 

soluble  protein,  fibrinogen,  into  the  insoluble  protein,  fibrin. 
Although  there  is  still  considerable  difference  of  opinion,  the 
mechanism  of  clotting  is  essentially  as  follows:  The  soluble 
fibrinogen  is  transformed  into  the  insoluble  fibrin  through  the 
agency  of  the  enzyme,  thrombin.  The  fibrinogen  exists  pre- 
formed in  the  plasma,  whereas  the  thrombin  is  derived  from  a 
precursor  substance — prothrombin;  and  for  the  conversion  of 
prothrombin  to  thrombin  the  presence  of  calcium  salts  is  essen- 
tial.1 As  may  be  observed  from  the  above  outline,  the  fluid  re- 
maining after  the  precipitation  of  fibrinogen  as  fibrin  is  serum. 
The  chemical  composition  of  the  blood  serum  is  fairly  con- 
stant, although  in  pathological  conditions  certain  more  or  less 
definite  variations  from  the  normal  may  be  encountered. 

PARTIAL    COMPOSITION    OF    BLOOD    SERUM.2 

Per  cent. 

Total  protein 7.0 

Globulin 2.7 

Albumin 4.3 

Incoagulable  and  non-protein  nitrogen 0 .  035 

Ash 0.88 

Chlorides 0 .36 

As  may  be  calculated  from  the  above,  the  globulin  makes  up 
about  38 .5  per  cent,  and  the  albumin  61 .5  per  cent,  of  the  total 
protein,  yielding  a  ratio  of  globulin  to  albumin  of  1:1.6; 
In  the  light  of  Epstein's  recent  studies  this  ratio  with  its  patho- 
logical variations  is  of  considerable  interest.  Epstein  found  that 
in  certain  diseases  the  proportion  of  globulin  is  much  higher, 
although  the  total  protein  of  the  serum  may  be  normal  or  even 
■reduced.  Thus  he  noted  an  increase  in  the  globulin  fraction  in 
"  (1)  cardiac  diseases  associated  with  decompensation  and  serous 
effusions,  (2)  pulmonary  or  respiratory  affections  ot  inflammatory 
or  non-inflammatory  origin  (pneumonia,  emphysema,  poly- 
cythemia), (3)  diabetes  mellitus,  and  (4)  parenchymatous 
nephritis."     In  fact,  in  cases  of  parenchymatous  nephritis  the 


1 .  Foi  further  discussion  of  the  mechanism  of  clotting  and  the  methods 
by  which  it  may  be  hastened  or  delayed,  see  various  papers  by  Howell 
and  his  co-workers,  also  Howell:  Text-book  of  Physiology,  New  York 
and  London,   1912. 

2.  Taken  from  Epstein:     Jour.  Exper.  Med.,  1912,  XVI,  p.  720. 


116  PATHOLOGICAL  CHEMISTRY 

globulin  was  found  to  make  up  as  much  as  95  per  cent,  of  the 
total  protein.  This  observation  may  be  of  interest  in  connection 
with  the  large  amounts  of  albumin  generally  present  in  the 
urine  in  this  condition. 

The  globulin  fraction  was  found  normal  or  reduced  in  achylia 
gastrica,  tuberculosis,  diabetes  insipidus,  and  chronic  interstitial 
nephritis.  Epstein  further  states  that  in  those  diseases  associated 
with  a  relatively  high  content  of  globulin  in  the  serum,  there 
occurs  an  accumulation  of  water  and  salts.  However,  there 
are  variations  which  cannot  be  accurately  interpreted. 

The  concentration  of  the  mineral  constituents  and  non-protein 
nitrogen  constituents  (digestive  and  metabolic  products)  of  the 
blood  is  assuming  considerable  importance  at  present  as  an  index 
to  the  efficiency  of  the  kidney  as  an  excretory  organ.  Folin 
and  Denis1  reported  that  for  a  series  of  16  normal  individuals  the 
non-protein  nitrogen  varied  between  22  and  26  milligrams  per 
100  grams  of  blood,  11  to  13  milligrams  per  100  grams  of  blood 
being  in  the  form  of  urea.  They  also  found  uric  acid  present  to 
the  extent  of  1  to  2  milligrams  per  100  grams  of  blood.  A 
marked  increase  in  urea  and  total  non-protein  nitrogen  was 
observed  in  syphilitics  and  insane  patients,  indicating  some  renal 
inefficiency.  In  cases  of  recognized  chronic  nephritis  the  total 
non-protein  nitrogen  rose  as  high  as  96  milligrams  per  100  grams 
of  blood  and  the  urea  nitrogen  68  milligrams  per  100  grams  of 
blood.  These  cases  are  not  necessarily  associated  with  an 
increased  concentration  of  uric  acid.  On  the  other  hand,  in 
many  cases  of  gout  the  uric  acid  may  accumulate  in  the  blood 
to  four  or  five  times  its  original  concentration  and  yet  be  ac- 
companied by  no  marked  retention  of  urea.  In  such  cases  the 
kidney  is  apparently  inefficient  only  in  so  far  as  the  elimination 
of  uric  acid  is  concerned. 

As  has  been  stated  in  Chapter  V,  in  connection  with  a  consid- 
eration of  diabetes,  blood  normally  contains  approximately  0 . 1 
per  cent,  dextrose,  which  may  experience  certain  changes  under 
special  conditions  as  there  outlined.  Reference  may  be  made  to 
this  chapter  for  cases  in  which  a  sugar  determination  may  be 
desirable. 

The  color  of  the  blood  is  due  to  the  hemoglobin  or  oxyhemo- 

1.  Folin  and  Denis:  Jour.  Biol.  Chem.,  1913,  XIV,  p.  29;  see  also 
Folin,  Karsner  and  Denis:     Jour.  Exper.  Med.,  1912,  XVI,  p.  789. 


CEREBROSPINAL   FLUID  117 

globin  contained  in  the  red  blood  corpuscles.  This  substance 
belongs  to  the  group  of  combined  proteins,  and  is  a  combination 
of  a  protein  (globin)  and  an  iron  bearing  coloring  matter  (hemo- 
chromogen),  which  is  readily  oxidized  to  hematin.  Hemoglobin 
unites  with  oxygen  in  the  lungs,  forming  oxyhemoglobin.  This 
combination  is  a  loose  one,  and  while  circulating  in  the  capillaries 
through  the  tissues  the  oxygen  is  given  up  and  reduced  hemo- 
globin again  formed.  Hemoglobin  is  thus  essentially  an  oxygen 
carrier  and  its  ability  to  take  up  oxygen  seems  to  be  a  function 
of  the  iron  it  contains.  Venous  blood  owes  its  characteristic 
purple  color  to  reduced  hemoglobin,  and  after  asphyxiation  this 
substance  is  practically  the  only  blood  coloring  matter  present. 
Under  certain  conditions  oxygen  may  become  more  firmly 
united  to  hemoglobin  than  is  the  case  in  oxyhemoglobin.  The 
compound  thus  formed  is  met  hemoglobin,  and  can  be  detected 
in  blood  after  poisoning  with  chlorates,  nitrites,  acetanilide 
and  many  other  substances.  Methemoglobin  contains  just 
as  much  oxygen  as  does  oxyhemoglobin,  but  the  oxygen  is 
given  up  much  less  readily,  and  hence  methemoglobin  cannot 
replace  oxyhemoglobin  as  an  oxygen  carrier.  Other  gases 
beside  oxygen  may  combine  with  hemoglobin,  e.g.,  carbon 
monoxide,  carbon  dioxide,  nitric  oxide.  Carbon-monoxide 
hemoglobin  and  nitric-oxide  hemoglobin  are  very  stable  com- 
pounds, and  of  course  cannot  function  as  oxygen  carriers. 

Cerebrospinal  Fluid. — There  appears  to  be  little  difficulty  in 
obtaining  sufficient  fluid  for  examination.  Blatteis  and  Lederer1 
succeeded  in  removing  fluid  in  all  of  their  426  cases,  and  Myers2 
states  that  25  cc.  of  fluid  were  easily  procured.  After  death, 
Myers  was  able  to  obtain  large  amounts  of  fluid — in  one  case 
over  200  cc.  Normally,  cerebrospinal  fluid  is  clear  and  colorless, 
with  a  specific  gravity  of  1 .  005-1 .  008  and  has  a  faintly  alkaline 
reaction.  Clear  and  colorless  fluids  are  also  observed  in  tuber- 
culous meningitis,  serous  meningitis,  hydrocephalus,  tumors  of 
the  brain  and  various  forms  of  mental  disturbances.  In  the 
last  mentioned  cases,  post  mortem  examinations  usually  reveal 
turbid  fluids.  Cloudy  fluids  are  indicative  of  acute  inflammatory 
processes.3     The  protein  of  cerebrospinal  > fluid  is  said  to  be,  for 


1.  Blatteis  and  Lederer:     Jour.  Amer.  Med.  Assoc,  1913,  LX,  p.  811. 

2.  Myers:     Jour.  Biol.  Chem.,  1909,  VI,  p.  123. 

3.  Cf.  Blatteis  and  Lederer:     loc.  cit. 


118  PATHOLOGICAL  CHEMISTRY 

the  most  part,  a  globulin,  seralbumin  being  present  only  under 
exceptional  conditions.  Normally,  the  protein  varies  from  0.02 
to  0.16  per  cent.,  and  is  increased  in  syphilitic  affections  and 
tubercular  meningitis,  where  it  may  reach  as  much  as  0.3  per 
cent.  Glucose  is  regularly  observed  in  cerebrospinal  fluid,  and 
choline  has  been  detected  in  several  diseases,  especially  in 
dementia  paralytica.1 

Transudates  and  Exudates. — Normally,  the  serous  membranes 
are  moistened  by  fluids,  which  (except  in  the  pericardial  sac)  are 
present  in  amounts  insufficient  for  complete  chemical  analyses. 
In  certain  pathological  states,  considerable  transudation  may 
take  place  from  the  blood  into  the  serous  cavities,  also  into  the 
subcutaneous  tissues  and  under  the  epidermis.  True  transudates 
are,  as  a  rule,  poor  in  cellular  elements  and  contain  relatively  little 
protein,  while  the  transudates  of  inflammatory  origin  (the  so- 
called  exudates)  are  rich  in  leucocytes  and  yield  more  protein. 
The  specific  gravity  of  transudates  varies  between  1 .  005  and 
1 .015,  and  their  protein  content  ranges  from  1  to  2.5  per  cent.; 
the  specific  gravity  of  exudates,  on  the  other  hand,  may  reach 
1 .  030  and  their  protein  concentration  4  to  6  per  cent.  In  general, 
transudates  and  exudates  are  clear  and  present  a  light  straw 
color.  Occasionally  an  admixture  of  blood  gives  them  a  reddish 
tinge,  in  which  case  they  are  said  to  be  hemorrhagic.  Ascitic 
fluid  may,  through  a  rupture  of  a  chylous  vessel,  become  rich  in 
finely  emulsified  fat,  although  ascitic  fluid  has  been  noted  to 
have  a  chylous  appearance  without  the  presence  of  fat.2 

LABORATORY    PROCEDURES. 

The  laboratory  methods  which  are  described  below  have 
been  included,  not  because  of  any  simplicity,  but  because  the 
data  obtained  with  them  may  prove  of  positive  diagnostic  value, 
and  have  already  yielded  data  of  scientific  interest.  Numerous 
other  determinations  might  be  described,  such  as  the  estimation 
of  hemoglobin,  the  specific  gravity,  lowering  of  the  freezing 
point,  etc.  The  estimation  of  hemoglobin  belongs  more  properly 
to  the  subject  of  hematology,  while  the  other  tests  may  be 

1.  For  further  discussion  on  cerebrospinal  fluid,  see  Myers:  loc.  cit.'* 
also  Simon:    Clinical  Diagnosis,  Philadelphia  and  New  York,  1911,  p.  474. 

2.  For  further  discussion  of  transudates  and  exudates  see  Simon: 
loc.  cit.,  p.  456;  also  Hammarsten-Mandel:  Text- book  of  Physiological 
Chemistry.    6th  Ed.     New  York,  1911,  p.  334.    - 


BLOOD   ANALYSIS  119 

found  in  any  text-book  on  the  subject,  and  have  not  been  shown 
to  be  of  any  definite  diagnostic  value. 

1 .  Sugar1. — From  15  to  25  grams  of  fresh  blood  are  transferred 
to  a  casserole  containing  about  150  cc.  of  phosphotungstic  acid 
solution.2  The  weight  of  the  blood  sample  is  conveniently 
obtained  by  weighing  the  casserole  with  the  phosphotungstic 
acid  before  and  after  the  addition  of  the  blood.  The  mixture  is 
thoroughly  stirred  and  boiled  until  the  protein  coagula  coalesce 
into  a  single  mass.  The  fluid  is  then  decanted  through  a  folded 
filter  paper  into  a  large  casserole.  Cold  water  is  added  to  the 
mass  of  coagulum  remaining  in  the  original  casserole,-  as  this 
treatment  renders  the  material  brittle,  and  enables  one  to  grind 
it  with  a  pestel  into  small  particles.  In  this  condition  the  coagu- 
lum can  be  easily  extracted  with  water,  three  successive  extrac- 
tions being  usually  sufficient  to  remove  all  the  sugar.  The  clear 
filtrate  with  washings  are  treated  with  10  per  cent.  NaOH  until 
only  a  very  faint  acid  reaction  remains.  This  nearly  neutralized 
solution  is  then  evaporated  on  the  water  bath  to  about  50  cc, 
transferred  to  a  100  cc.  volumetric  flask  and  made  up  to  the  100 
cc.  mark  with  water.  The  sugar  is  then  determined  by  means 
of  Allihn's  gravimetric  method,  employing  the  two  component 
solutions  contained  in  Fehling's  solution.3  Place  30  cc.  of  the 
copper  solution,  30  cc.  of  the  alkaline  tartrate  solution  and  60 
cc.  of  water  in  a  casserole  of  medium  size,  and  heat  to  boiling. 
25  cc.  of  the  sugar  solution  are  then  added  and  the  mixtures 
again  brought  to  a  boil  and  maintained  at  the  boiling  point  for 
exactly  two  minutes.  At  the  end  of  this  interval  the  mixture  is 
immediately  filtered  by  means  of  suction  through  a  weighed 
Gooch  crucible  containing  asbestos,  the  red  precipitate  of 
cuprous  oxide  being  transferred  quantitatively  to  the  crucible 
with  the  aid  of  hot  water  and  a  rubber  tipped  glass  stirring  rod. 
Alcohol  is  then  added  to  facilitate  drying'  and  the  crucible 
.  carefully  heated  in  the  upper  part  of  the  flame  of  a  Bunsen 
burner.  This  ignition  converts  the  red  cuprous  oxide  into  the 
black  cupric  oxide,  ten  minutes  of  heating  usually  being  sufficient. 

1.  Bang  has  recently  proposed  methods  for  the  estimation  of  sugar, 
also  moisture,  protein  and  chlorides,  which  require  as  little  as  0. 1  gram 
of  blood.  These  microchemical  methods  described  by  Bang  are 
apparently  capable  of  yielding  reliable  data.  See  Biochem.  Zeitschr., 
1913,  XLIX,  p.  19. 

2.  See  foot-note  Chapter  IX,  p.  112. 

3.  See  foot-note  Chapter  V,  p.  75. 


120  •  PATHOLOGICAL  CHEMISTRY 

The  crucible  is  cooled  and  weighed.  The  difference  between  this 
weight  and  the  original  weight  of  the  crucible  represents  the 
weight  of  cupric  oxide,  which  should  be  multiplied  by  0.9 
to  obtain  its  equivalent  in  terms  of  cuprous  oxide.  The  amount 
of  dextrose  corresponding  to  this  may  be  obtained  from  the 
tables  published  in  the  "  Official  and  Provisional  Methods  of 
Analysis,"  pp.  50-51. *  Multiplying  this  value  by  4  gives  the 
amount  of  sugar  present  in  the  sample  of  blood  taken  for  analysis, 
from  which  the  percentage  concentration  may  be  readily  cal- 
culated. 

Less  accurately  the  sugar  in  the  peripheral  blood  stream  may 
be  estimated  by  collecting  5  cc.  of  blood,  and  at  once  mixing  with 
100  cc.  of  absolute  alcohol,  filtering  off  the  precipitate  and  wash- 
ing with  a  little  hot  95  per  cent,  alcohol.  The  filtrate  is  evaporated 
nearly  to  dryness  in  a  small  evaporating  dish  on  the  water 
bath,  then  completely  transferred  to  a  still  smaller  dish,  washed 
with  a  little  hot  water,  and  evaporated  practically  to  dryness. 
This  is  now  completely  washed  with  5  one  tenth  cc.  portions 
of  boiling  water  into  a  Lohnstein  saccharimeter,  0.5  cc.  of  yeast 
emulsion  added,  etc.  as  described  in  Chapter  V.  Since  5  cc.  of 
blood  have  been  employed  the  reading  is  divided  by  10. 

2  .  Total  Non-protein  Nitrogen  (Folin- Denis)2. — For  the  deter- 
mination of  the  total  non-protein  nitrogen  and  also  the  urea  in 
human  blood,  5  cc.  of  blood  are  necessary.  Folin  and  Denis 
suggest  the  following  method  for  drawing  the  blood :  An  ordinary 
5  cc.  pipette  is  connected  to  a  small  sterile  hypodermic  needle 
with  the  aid  of  a  short  piece  of  pure  gum  tubing.  The  pipette 
should  be  perfectly  dry  and  into  the  upper  end  is  introduced  a 
pinch  of  powdered  potassium  oxalate  which  is  allowed  to  run 
down  into  the  tip.  The  upper  end  is  then  connected  with  a 
rubber  tube  having  a  pinch  cock.  In  this  way  exactly  5  cc. 
of  blood  may  be  drawn  without  clotting.  The  blood  is  then 
transferred  at  once  to  a  50  cc.  volumetric  flask  half  filled  with 
methyl  alcohol  (acetone  free).  The  flask  is  then  filled  up  to  the 
mark  and  vigorously  shaken.  Sometime  after  two  hours  the 
contents  of  the  flask  is  filtered  through  a  dry  filter.  To  the 
filtrate  is  then  added  2  to  3  drops  of  a  saturated  alcoholic  solution 
of  zinc  chloride,  and  after  standing  for  a  few  minutes,  the  mixture 

1.  This  publication  (Bulletin  No.  107  revised)  may  be  obtained 
from  the  U.  S.  Department  of  Agriculture,  Bureau  of  Chemistry. 

2.  Folin    and   Denis:     Jour.  Biol.  Chem.,  1912,  XI,  p.  527. 


BLOOD  ANALYSIS  121 

is  again  filtered  through  a  dry  paper.  To  determine  the  total  non- 
protein nitrogen  of  the  blood,  10  cc.  of  the  alcoholic  filtrate  are 
transferred  to  a  large  Jena  test  tube  (20-25  mm.  by  200  mm.). 
One  drop  of  sulphuric  acid,  one  of  kerosene  and  a  pebble  are 
added  and  the  methyl  alcohol  driven  off  by  immersing  the  test 
tube  in  a  beaker  of  boiling  water  for  five  to  ten  minutes.  When 
the  alcohol  is  removed,  1  cc.  of  concentrated  sulphuric  acid,  a 
gram  of  potassium  sulphate,1  and  a  drop  of  copper  sulphate 
are  added  and  boiled  over  a  micro-burner  for  about  two  minutes 
after  the  solution  becomes  colorless.  It  is  allowed  to  cool  for 
about  three  minutes  until  it  just  becomes  viscous,  then  diluted 
with  about  6  cc.  of  water,  adding  the  water  slowly  at  first,  then 
more  rapidly  to  prevent  solidification.  To  this  mixture,  after 
the  necessary  apparatus  has  been  adjusted,  is  added  an  excess 
of  sodium  hydroxide  (3  cc.  of  the  saturated  solution)  and  the 
ammonia  removed  either  by  aeration  with  compressed  air  or 
suction  as  described  by  Foiin  and  Denis,2  or  by  simple  distilla- 
ton  with  a  very  small  condenser  into  a  second  large  test  tube 
containing  1  cc.  of  N/10  acid  and  2-3  cc.  of  water,  as  in  the  usual 
Kjeldahl  distillation.  About  ten  minutes  usually  suffice.  The 
great  delicacy  of  the  method  as  described  by  Folin  and  Denis 
depends  upon  the  determination  of  the  ammonia  thus  obtained, 
with  the  aid  of  Nessler's  solution,  permitting  the  use  of  material 
containing  considerably  less  than  1  mg.  of  nitrogen.  For  this 
determination  in  human  blood  7  to  8  cc.  of  diluted  Nessler's 
reagent3  (diluted  1  to  5  just  previous  to  use)    are  added  and  the 

1.  The  necessity  of  having  ammonia  free  reagents  and  distilled 
water  is  obvious. 

2.  For  various  detailed  directions  in  this  connection  it  may  be 
necessary  to  consult  their  original  paper. 

3.  Nessler's  solution  is  prepared  by  dissolving  62.5  grams  of  potas- 
sium iodide  in  about  250  cc.  of  distilled  water,  setting  aside  a  few  cc.  and 
adding  gradually  to  the  larger  part  a  cold  saturated  solution  of  mercuric 
chloride  (or  mercuric  iodide),  of  which  about  500  cc.  will  be  required, 
until  the  mercuric  iodide  precipitated  ceases  to  be  redissolved  on  stirring. 
When  a  permanent  precipitate  is  obtained,  restore  the  reserved  potassium 
iodide  so  as  to  redissolve  it,  and  continue  adding  mercuric  chloride  very 
gradually  until  a  slight  precipitate  remains  undissolved.  Next  dissolve 
150  grams  of  pure  potassium  hydroxide  in  150  cc.  of  distilled  water,  cool, 
add  gradually  to  the  above  solution,  and  make  up  with  distilled  water 
to  one  liter.  On  standing,  a  brown  precipitate  is  deposited,  and  the 
solution  becomes  clear,  and  of  a  pale  greenish-yellow  color.  The  clear 
supernatant  fluid  is  decanted  into  a  smaller  bottle  as  required  for  use. 


122  PATHOLOGICAL    CHEMISTRY 

solution  carefully  washed  into  a  25  cc.  volumetric  flask  and 
made  up  to  volume.  (If  much  ammonia  is  present,  so  that  the 
resulting  colored  solution  must  be  diluted  to  50  or  100  cc, 
correspondingly  large  amounts  of  Nessler's  reagent  are  added.) 
Simultaneously  with  the  development  of  the  color  in  this  solution, 
1  mgm.  of  nitrogen  (as  ammonium  sulphate)  is  Nesslerized  in  a 
100  cc.  flask  with  25  cc.  of  the  diluted  reagent.  The  flask  is 
made  up  to  the  mark,  mixed,  and  a  portion  poured  into  one  of 
the  cups  of  the  colorimeter  and  the  prism  set  at  20  mm.  The 
comparison  is  now  made.  Since  the  equivalent  of  1  cc.  of  blood 
has  been  employed  the  calculation  is  very  simple. 

3.  Urea. — In  the  determination  of  urea,  10  cc.  of  the 
alcoholic  filtrate  are  again  employed.  This  is  carefully 
evaporated  to  dryness  in  a  similar  test  tube  after  the  addition 
of  a  drop  of  dilute  acetic  acid  and  two  or  three  of  kerosene. 
Folin  and  Denis  add  to  the  residue  2  cc.  of  25  per  cent,  acetic 
acid,  a  pebble  and  7  grams  of  dry  potassium  acetate,  and  the 
decomposition  is  carried  out  by  heating  to  153-158°C.  for  8  to  10 
min.,  using  a  calcium  chloride  tube  without  bulb  as  condenser. 
Benedict's  method  as  described  for  urine1  can  perhaps  be  em- 
ployed more  simply.  The  ammonia  is  finally  removed  after 
the  addition  of  2  cc.  of  saturated  sodium  hydroxide  in  a  similar 
manner  to  that  described  for  the  total  non-protein  nitrogen. 
In  this  case  10  cc.  volumetric  flasks  are  necessary  in  connection 
with  the  Nesslerization  for  which  3  cc.  of  the  dilute  reagent 
are  required.  As  only  10  cc.  of  solution  are  available,  a  dry 
colorimeter  cup  must  be  at  hand.  The  same  standard  and 
similar  calculations  to  the  above  are  employed. 

4.  Uric  Acid  •( Folin- Denis)2. — The  method  is  based  upon 
the  color  reaction  previously  employed  by  Folin  and  Macallum3 
for  the  estimation  of  uric  acid  in  urine.  For  the  determination, 
15  to  25  cc.  of  blood  are  employed.  The  blood  is  drawn  into  a 
small,  weighed,  wide-mouthed  bottle  or  test  tube  containing 
about  0 . 1  gram  of  powdered  potassium  oxalate.  The  weight  of 
the  blood  is  obtained  by  difference,  and  five  times  the  weight 
of  N/100  acetic  acid  is  heated  to  boiling  in  an  ordinary  liter 


1.  Chapter  III,  p.   45. 

2.  Folin     and     Denis:     Jour.   Biol.   Chem.,   1913,   XIII,  p.  469,.  also 
XIV,  p.  95. 

3.  Folin    and    Macallum:     Ibid.,  1912,  XIII,  p.  363. 


BLOOD   ANALYSIS  123 

flask,  the  oxalated  blood  added,  and  the  mixture  again  heated  to 
boiling.  The  mixture  is  filtered  while  still  hot.  The  coagulated 
blood  is  stirred  up  once  in  about  200  cc.  of  boiling  water,  allowed 
to  stand  about  5  minutes  and  filtered  through  the  same  filter. 
The  combined  filtrate  and  wash  water  containing  the  uric  acid 
and  other  soluble  materials  is  further  acidified  with  5  cc.  of  50 
per  cent,  acetic  acid  and  evaporated  in  a  suitable  porcelain  dish 
to  about  3  cc.  The  liquid  is  then  poured  into  a  15  cc.  centrifuge 
tube  and  the  dish  washed  with  two  successive  portions  of  0.1 
per  cent,  lithium  carbonate  solution,  using  about  2  cc.  for  each 
rinsing,  any  solid  material  being  removed  with  the  aid  of  a 
rubber- tipped  stirring  rod.  To  the  liquid  in  the  centrifuge  tube, 
which  should  not  be  greater  than  10  cc.  in  volume,  are  added  5 
drops  of  3  per  cent,  silver  lactate  solution,  2  drops  of  magnesia 
mixture  and  sufficient  strong  ammonium  hydroxide  (10  to  15 
drops)  to  dissolve  the  silver  chloride.  The  tube  is  centrifuged 
for  two  or  three  minutes,  the  supernatant  liquid  poured  off  and 
to  the  residue  are  added  4  to  5  drops  of  fresh  saturated  hydrogen 
sulphide  water  and  one  drop  of  concentrated  hydrochloric  acid. 
The  tube  is  now  placed  for  a  period  of  five  to  ten  minutes  in  a 
beaker  of  boiling  water  in  order  to  remove  the  excess  of  hydrogen 
sulphide.  Since  hydrogen  sulphide  produces  a  blue  color  reaction 
with  the  phosphotungstic  reagent,  it  is  necessary  to  remove  every 
trace  of  this  substance.  To  secure  this,  a  drop  of  0.5  per  cent, 
lead  acetate  is  added  to  the  contents  of  the  centrifuge  tube  as 
it  is  taken  out  of  the  hot  water.  (If  any  blackening  should  occur, 
the  tube  should  be  heated  for  another  five  minutes  and  another 
drop  of  lead  acetate  added.)  The  tube  is  now  centrifuged, 
the  supernatant  liquid  transferred  by  decanfation  as  completely 
as  possible  to  a  small  beaker  and  the  inside  of  the  tube  washed 
with  a  fine  stream  of  water,  care  being  taken  to  disturb  as  little 
as  possible  the  solid  residue  in  the  bottom  of  the  tube.  The  wash 
water  (should  not  exceed  5  cc.)  is  added  to  the  liquid  in  the 
beaker  and  to  this  acid  solution  containing  the  uric  acid  is  then 
added  2  cc.  of  the  uric  acid  reagent1  and  10,  15,  or  20  cc.  of 
saturated   sodium   carbonate   solution,   depending   on   whether 


1 .  The  reagent  is  prepared  by  boiling  100  grams  of  sodium  tungstate 
and  80  cc.  of  85  per  cent,  phosphoric  acid  in  750  cc.  of  distilled  water  for 
two  hours,  preferably  under  a  reflux  condenser,  and  then  making  up  to 
1000  cc.  with  water. 


124  PATHOLOGICAL  CHEMISTRY 

the  color  obtained  requires  a  final  dilution  to  25,  50,  or  100  cc. 
Flasks  of  these  capacities  should  be  at  hand,  and  the  blue  un- 
known solution  is  transferred  to  one  of  them  and  diluted  with 
water  to  the  mark.  Five  cc.  of  the  standard  uric  acid-formalde- 
hyde reagent1  are  placed  in  a  100  cc.  flask,  2  cc.  of  the  uric 
acid  reagent,  20  cc.  of  sodium  carbonate  added,  and  the  solution 
made  up  to  100  cc,  this  being  done  just  previously  to  the  addition 
of  sodium  carbonate  to  the  unknown.  The  latter  sometimes 
needs  to  be  filtered  before  being  transferred  to  the  colorimeter 
cylinders  for  the  final  color  comparison.  The  mm.  position  at 
which  the  standard  should  be  set  depends  upon  individual  con- 
ditions. Knowing  the  strength  of  the  standard  reagent  and  the 
amount  of  blood  employed,  the  calculation  of  the  uric  acid  per 
100  cc.  of  blood  is  not  difficult. 

The  laboratory  procedures  included  in  the  ordinary  routine 
physical  and  chemical  examination  of  cerebrospinal  fluid,2 
transudates,  exudates,  etc.  include  volume,  color,  specific  gravity, 
reaction,  sugar,  and  protein  content,  for  which  the  methods 
described  in  previous  chapters  will  serve.  Occasionally  some 
special  examination  is  required.  It  may  be  desired  to  learn  whether 
or  not  a  fluid  is  contaminated  with  urine,  and  for  this  purpose 
the  creatinine  reaction  is  appropriate,  as  it  is  only  in  urine  that 
a  pronounced  test  is  obtained. 

1 .  This  standard  is  prepared  by  dissolving  one  gram  of  uric  acid  in 
200  cc.  of  0.4  per  cent,  lithium  carbonate  in  a  liter  flask.  Solution  is 
quickly  brought  about  by  shaking  and  40  cc.  of  40  per  cent,  formaldehyde 
solution  are  added  and  the  mixture  allowed  to  stand  for  a  few  minutes. 
The  clear  solution  is  acidified  by  the  addition  of  20  cc.  of  normal  acetic 
acid  and  diluted  to  trje  liter  mark  with  water.  On  the  next  day,  it  is 
standardized  against  a  freshly  prepared  lithium  carbonate  solution  of 
uric  acid.  The  color  produced  by  5  cc.  of  this  solution  corresponds  very 
nearly  with  that  obtained  with  1  mg.  of  uric  acid.  This  standard  appears 
to  keep  permanently. 

2 .  For  a  consideration  of  the  use  of  lumbar  puncture  in  children  see 
Pisek:     Post-Graduate,  1912,  XXVII,  p.  892. 


APPENDIX. 
Laboratory  Suggestions. 

The  laboratory  suggestions  to  follow  are  intended  primarily  for 
individuals  comparatively  unfamiliar  with  the  technique  of  the 
various  laboratory  manipulations  The  simple  forms  of  ap- 
paratus which  have  been  found  of  service  in  the  tests  previously 
described  are  in  part  shown  in  the  accompanying  illustration. 
With   a  few  exceptions  they  are  inexpensive. 

Apparatus. — The  uses  of  the  various  pieces  of  apparatus 
marked  in  Fig.  13  may  be  briefly  given  as  follows: 

A ,  screw-top  jar,  suitable  tor  the  collection  of  gastric  contents 
and  feces. 

B,  urinary  sedimenting  glass  with  funnel,  convenient  for  the 
filtration  of  gastric  contents. 

C,  porcelain  evaporating  dish,  with  50  cc.  burette  and  stand, 
used  for  the  titration  of  gastric  acidities,  and  chlorides  and 
phosphates  of  urine.  The  burette  is  also  employed  for  the 
titration  of  urinary  acidity  and  ammonia  in  connection  with  the 
Erlenmeyer  flask  K.  The  burette,  as  here  illustrated,  has  a  rubber 
pinchcock.  This  is  to  be  preferred  to  a  glass  stopcock  for  N/10 
NaOH  in  clinical  work,  as  glass  stopcocks  are  very  likely  to  stick 
after  having  been  used  with  alkali,  unless  very  carefully  cleaned. 

D,  small  test  tube  rack  with  small  test  tubes,  suitable  for  the 
estimation  of  pepsin  (Rose's  Method),  amylase  in  duodenal  juice, 
etc. 

E,  small  mortar,  employed  in  preparing  feces  for  micro- 
chemical  examination. 

Fj  fermentation  tube  for  feces,  showing  vessel  a,  and  tubes  b 
and  c  as  described  in  Chapter  II. 

G,  test-tube  rack  designed  for  urine  work.  The  rack  as  here 
illustrated  has  places  for  three  rows  of  test  tubes,  the  lower  for 
large  tubes  (1  inch  in  diameter)  for  observation  of  color,  odor, 
reaction  and  sp.  gr.,  and  from  which  samples  of  urine  may  be 
withdrawn  for  the  various  qualitative  tests.  The  middle  row 
holds  the  tubes  used  for  the  sugar  test,  and  the  upper  row,  a  set 

125 


126  PATHOLOGICAL  CHEMISTRY 

of  small  tubes  for  Heller's  cold  nitric  acid  test  for  albumin.  With 
this  rack,  a  series  of  urines  may  be  examined  very  rapidly 
without  confusion.  Where  a  large  number  of  urines  are  to  be 
examined,  as  in  hospital  work,  a  series  of  racks  for  12,  instead  of  6 
tubes  may  be  employed.1 

H,  apparatus  employed  for  Benedict  sugar  titration,  consists, 
of  Bunsen  burner,  tripod  with  wire  gauze,  and  25  cc.  burette 
with  stand.  The  25  cc.  burette  with  glass  stopcock  is  convenient, 
although  the  50  cc.  burette  might  be  employed.  Where  one 
burette  is  to  be  used  for  all  determinations,  the  25  cc.  instrument 
as  here  illustrated  is  preferable.  It  should  then  always  be  care- 
fully washed  out  after  use,  care  being  taken  to  see  that  the  stop- 
cock is  covered  with  a  thin  coat  of  vaseline. 

/,   improved    Lohnstein   saccharimeter. 

J,  Esbach's  albuminometer. 

K,  Erlenmeyer  flask,  suitable  for  titrating  urinary  acidity  and 
ammonia. 

L,  liter  and  two-liter  cylinders  for  making  up  solutions  and 
measuring  volume  of  urine. 

M,  Duboscq  colorimeter  employed  in  estimating  phenol  - 
sulphonephthalein,  creatinine,  indican,  and  many  other  sub- 
stances. For  phenolsulphonephthalein,  the  Hellige  instrument, 
which  costs  one-fourth  as  much  as  the  Duboscq,  gives  satis- 
factory results. 

iV,  Leitz  microscope,  without  oil  immersion  lens,  but  with 
condenser  and  iris  diaphragm.  Very  suitable  for  work  in  tests 
previously  described,  and  comparatively  inexpensive. 

0.  cylinder  with  filter  paper  in  bottom  containing  a  series  of 
pipettes,  1  cc.  graduated  in  1/100,  1  cc,  5  cc,  10  cc,  25  cc,  and 
50  cc,  and  covering  all  the  different  quantitative  methods. 

P,  set  of  dropping  bottles,  containing  various  indicators, 
phenolphthalein,  dimethylaminoazobenzene  and  alizarin. 

Q,  set  of  reagent  bottles,  permanently  labeled,  very  convenient, 
though  not  essential. 

When  it  is  necessary  to  make  a  large  number  of  qualitative 
tests  for  sugar  in  urine,  as  is  generally  the  case  in  routine  hospital 
work,  the  apparatus  illustrated  in  Fig.  14,2  and  employed  in 
connection  with  Benedict's  qualitative  solution  will  be  found 

1.  Cf.  Myers:      New  York  Med.  Jour.,  1913,  XCVII,  p.  1126. 

2.  Myers:  loc.  cit. 


Fig.  14 


LABORATORY  SUGGESTIONS  127 

to  greatly  reduce  the  time  necessary  to  make  the  tests  without 
sacrifice  to  their  accuracy.  A  series  of  tubes  containing  Benedict's 
solution,  to  which  the  eight  drops  of  urine  have  been  added  and 
the  tubes  agitated,  are  placed  in  their  numerical  order  in  the 
numbered  places  of  a  special  copper  rack.  The  rack  is  then 
immersed  below  the  upper  level  of  the  Benedict  solution  for  two 
minutes  in  a  bath  of  saturated  calcium  chloride,  which  has  just 
been  brought  to  the  boiling  point  and  the  flame  removed.  At 
the  end  of  the  two  minutes  the  rack  is  elevated,  the  tubes  al- 
lowed to  drain  and  cool,  and  any  tubes  which  show  a  positive 
reaction  noted. 

Form  of  Reports  and  Tests  Usually  Included. — Below  are  tabu- 
lated the  forms  of  report  blanks  usually  employed  m  the  exami- 
nation of  various  specimens  for  diagnostic  purposes.  In  the  first 
column  are  found  the  tests  which  comprise  the  routine  ex- 
amination, unless  otherwise  noted.  This  scheme  is  convenient 
for  a  card  index  file. 

EXAMINATION  OF  GASTRIC  CONTENTS 

Name  of  Patient Date Age Sex 

Test  Meal Microscopical  Examination 

Time  Taken Erythrocytes 

Time  Expressed Leucocytes 

Color Bacteria 

Consistency Yeasts 

Odor Special  Examination 

Mucus Blood  (chemical) 

Quantity. . Pepsin 


Sediment 

Character Rennin. 


,  Total Peptone. 

Volume 


Filtrate. Starch  Products.  . 

Chemical  Examination  Tryptophan  Test. 

Salomon's  Test. . . 
(  Free Remarks 


(  Combined. 


Total  Acidity 

Lactic  Acid 

The  usual  routine  in  the  examination  of  gastric  contents 
(after  the  Ewald  Meal),  as  noted  above,  includes  the  determina- 
tion of  the  acidities,  especially  free  HC1  and  the  total  acidity, 
also  the  test  for  lactic  acid  in  the  absence  of  free  HC1.  The 
microscopical  examination,  the  test  for  V  occult  "  blood,  the 


128  PATHOLOGICAL  CHEMISTRY 

estimation  of  pepsin,  the  tryptophan  test  and  the  Salomon  test 
come  into  consideration  in  special  conditions,  such  as,  e.g., 
carcinoma  of  the  stomach. 

EXAMINATION  OF  FECES 

Name  of  Patient Date Age Sex 

Macroscopic  Examination  Special  Chemical  Examination 

Color „ Blood 

Odor Fermentation  Test 

Consistency Schmidt's  Reaction 

Reaction Albumin 

Mucus Sugar 

Microscopical  Examination 

Connective  Tissue Fat 

Mucus Remarks 

Muscle  Fiber 

(  Globules • 


Fat     < 

j  (  Crystals. 


Starch 

Erythrocytes 

Leucocytes. . 

Parasites 

The  macroscopic  examination  of  feces  when  properly  made  is 
of  very  great  clinical  value,  often  enabling  the  experienced  ob- 
server to  form  a  diagnosis.  The  microscopical  examination  is  of 
especial  value  after  the  intestinal  test  diet,  as  indicated  in  Chapter 
II.  Of  the  special  chemical  tests,  that  for  "  occult  "  blood  is  the 
most  important  clinically,  though  here  it  is  important  that  the 
diet  from  which  the  feces  are  obtained  should  be  free  from  meat. 

EXAMINATION  OF  URINE 

Name  of  Patient Date Age Sex 

Chemical  Examination  Special  Chemical  Examination 

Volume  in  24  hrs Bile 

Specific  Gravity Blood 

Color Indican ....". 

Odor Chlorides  as  NaCl 

Reaction Total  Nitrogen 

Sediment Ammonia 

Albumin Urea 

Sugar Creatinine 

Acetone Uric  Acid 

Diacetic  Acid Phenolsulphonephthalein 


LABORATORY  SUGGESTIONS  129 

Microscopical  Examination 

Remarks 


The  usual  clinical  examination  of  urine  includes  the  list  under 
chemical  examination  and  microscopical  examination  above, 
except  the  tests  for  acetone  and  diacetic  acid.  * 

The  tests  for  acetone  and  diacetic  acid  should  always  be 
included  when  sugar  has  been  found  to  be  present.  Sugar  when 
detected  should  be  estimated,  not  simply  the  percentage  elimina- 
tion determined,  but  the  grams  of  glucose  excreted  in  twenty- 
four  hours.  If  an  acidosis  is  present  (diabetes,  pernicious  vomit- 
ing of  pregnancy,  eclampsia,  etc.),  the  degree  of  this  can  very 
easily  be  gauged  by  the  ammonia  elimination. 

The  quantitative  estimation  of  albumin  is  often  of  value  in 
cases  of  albuminuria,  although  the  quantitative  data  are  not  as 
important  here  as"  in  glucosuria. 

Under  the  heading  of  special  chemical  examinations,  in  addition 
to  ammonia,  those  clinically  the  most  important  are  indican, 
chlorides,  total  nitrogen  and  phenolsulphonephthalein.  The 
indican  is  often  an  index  as  to  the  amount  of  a  certain  type  of 
intestinal  putrefaction.  The  chloride  estimation  is  of  value  in 
pneumonia  and  certain  cases  of  edema.  Where  it  is  desired  to 
ascertain  the  ability  of  the  body  to  eliminate  organic  material, 
the  total  nitrogen  elimination  is  much  to  be  preferred  to  the  old 
urea  estimation,  though  it  is  obvious  that  a  knowledge  of  the 
nitrogen  content  of  the  diet  is  a  prerequisite.  In  the  cases  where 
it  is  desired  to  test  the  functional  capacity  of  the  kidney,  the 
phenolsulphonephthalein  test  may  be  of  great  value. 

Recording  the  examination  of  milk  or  other  fluids  is  required 
of  the  physician  so  infrequently  that  no  special  mention  of 
them  will  be  made  here. 

Uniform  Charting  of  Qualitative  Tests. — For  the  sake  of  uni- 
formity, .it  is  very  important  to  employ  a  systematic  scheme  in 
charting  all  qualitative,  reactions,  viz.,  lactic  acid  in  gastric 
contents,  "  occult  "  blood  in  gastrics  and  feces,  albumin,  sugar, 
acetone,  diacetic  acid  and  indican  in  urine,  etc.  The  following 
has   been   found   convenient,  faint  trace,   trace,   small  amount, 


130  PATHOLOGICAL  CHEMISTRY 

moderate  amount,  large  amount,  and  very  large  amount.  For 
microscopical  work,  especially  in  the  examination  of  urinary 
sediments,  the  following  scheme,  which  will  apply  to  both  or- 
ganized and  crystalline  sediments,  may  be  employed — an  occa- 
sional (hyaline  cast),  a  few  (uric  acid  crystals),  a  moderate 
number  (of|pus  cells),  many  (finely  granular  casts),  very  many 
(calcium  oxalate  crystals). 


CONVENIENT  REFERENCE  BOOKS. 
When    more   detailed   information   is  desired    on    the    various    topics 
presented  in  the  preceding  chapters,  the  experience  of  the  authors  has 
shown  the  following  books  to  be  particularly  useful. 

For  the  general  application  of  chemical  findings  to  diagnosis: 

Simon:     Clinical  Diagnosis. 

Wood:     Chemical  and  Microscopical  Diagnosis. 
For  the  presentation  of  various  phases  of  digestion  and  nutrition: 

Howell:     Text-book  of  Physiology. 

Hammarsten-Mandel;    Text-book  of  Physiological  Chemistry. 

Lusk:     Science  of  Nutrition. 

Sherman:     The  Chemistry  of  Food  and  Nutrition. 

Taylor:     Digestion  and  Metabolism. 

Krehl- Hewlett:     Clinical  Pathology. 

von  Noorden-Hall:     Metabolism  and  Practical  Medicine. 
For  a  discussion  of  the  diseases  of  the  stomach  and  intestines,  and  of  the 
composition  of  the  feces: 

Cohnheim  (P.) — Fulton:     Diseases  of  the  Digestive  Canal. 

Einhorn:     Diseases  of  the  Stomach. 

Einhorn:     Diseases  of  the  Intestines. 

Schmidt- Aaron:    Test  Diet  in  Intestinal  Diseases. 

Schmidt  und  Strasburger:     Die  Fazes  des  Menchen. 
For  further  details  on  modern  quantitative  methods  of  urine  analysis: 

Hawk:     Practical  Physiological  Chemistry. 
For  a  discussion  of  diabetes: 

Cammidge:     Glycosuria  and  Allied  Conditions. 

von  Noorden:     New  Aspects  of  Diabetes. 
For  a  consideration  of  milk  and  its  relation  to  infant  nutrition: 

Langstein  und   Meyer:     Sauglingsernahrung  und   SauglingsstofT- 

wechsel. 


INDEX. 


Acetone,  79. 

determination  of,  86. 

origin  of,  80. 

tests  for,  84. 
Acetone  bodies,  79. 

relation  to  ammonia  elimination, 
82. 

relation  to  coma,  81,  82. 
Acidosis,  79-87. 

after  anesthesia,  83. 

in  cyclic  vomiting  in  children,  83. 

in  diabetes,  80,  81. 

in  eclampsia,  83. 

in  febrile  diseases,  83,  84. 

in  pernicious  vomiting  of  preg- 
nancy, 83. 

of  protein-fat  diet,  80. 

of  starvation,  80. 

use  of  alkali  in,  82,  83. 
Acid,  boric,  113. 

diacetic,  79,  80,  84,  86. 

fatty,  12,  17,  79,  81. 

hippuric,  41. 

homogentisic,  52,  88. 

/3-hydroxybutyric,  79,  80,  85. 

hydrochloric,  1,  2,  3,  5. 

indoleacetic,  91,  95. 

lactic,  3,  7,  64,  83. 

oxalic,  41,  100. 

volatile  fatty,  3. 

uric,  36,  47,  116,  122. 
Adrenals,  66. 
Albumin,  in  urine,  49,  50. 

tests  for,  54. 

quantitative  estimation  of,  55. 
Albuminuria,  49-59. 

due  to  circulatory  disturbances, 
50. 

due  to  toxic  substances,  50. 

in  febrile  conditions,  50. 

in  acute  nephritis,  50. 


Albuminuria,  in  chronic  parenchy- 
matous nephritis,  51. 
in   chronic  interstitial  nephritis, 

51. 
in  amyloid  disease,  51. 
Alkaptonuria,  52. 

Amino   acids,    12,    13,    35,    51,    58, 
70,  83. 
estimation  of,  58. 
in  digestion,  12. 
in  the  blood,  13. 
in  the  urine,  51. 
Ammonia,  as  an  index  of  acidosis, 
82. 
estimation  of,  43. 
normal  and  abnormal    excretion 
of,  34,  82. 
Ammonium  magnesium  phosphate, 

100. 
Ammonium  urate,  101. 
Apparatus,  125-127. 
Arabinose,  see  pentose. 

Bacteria,  in  feces,  14,  16. 

in  gastric  contents,  3,  11. 

in  urine,  96,  99. 
Bence-Jones'  protein,  51. 
Bile  pigments,  in  feces,  16,  22. 

in  urine,  89. 

tests  for,  92. 
Blood,  114-124. 

composition  of,  114. 

in  feces,  23. 

in  gastric  contents,  9,  11. 

in  urine,  90,  98. 

non-protein  nitrogen  of ,  116,  120, 
121. 

pigments  of,  117. 

protein  of,  115. 

sugar  in,  60,  116,  119. 

tests  for,  23,  93. 


131 


132 


INDEX. 


Blood,  urea  in,  116,  122. 
uric  acid  in,  116,  122. 
Boas-Oppler  bacillus,  3. 
Boric  acid,  detection  in  milk,  113. 

Calcium,  in  urine,  33. 
Calcium  carbonate,  in  urinary  sedi- 
ments, 100. 
Calcium   phosphate,   in  milk,    105. 

in  urinary  calculi,  101,  102. 

in  urinary  sediments,  100. 
Calcium  oxalate,  in  urinary  calculi, 
101. 

in  urinary  sediments,  100. 
Calculi,  biliary,  21,  24. 

urinary,  101,  102. 
Carbohydrates,  digestion  of,  12. 

in  blood,  60,  68,  119. 

in  milk,  105,  112. 

in  urine,  60. 

requirement  of,  1. 

utilization  of,  18. 
Casein,  in  milk,  104,  108.( 
Casts,  urinary,  96,  97. 
Cerebrospinal  fluid,  117,  124. 
Chlorides,  determination  of.,  42. 

elimination  of,  32,  53,  58. 

in  blood,  114. 

in  milk,  105. 
Colostrum,  108. 
Creatine,  determination  of,  48. 

elimination     of     in     pathological 
conditions,  40. 

origin  of,  40. 
Creatinine,  determination  of,  47. 

elimination  of,  39. 

origin  of,  38. 
Cylindroids,  97. 

Cystine,  in  urinary  sediments,  101. 
Cystinuria,  52. 

Dextrose,  see  glucose. 
Diabetes  insipidus,  29. 
Diabetes  mellitus,  classification  of, 
67. 

G:N  ratio,  70. 

protein  metabolism  in,  69. 

respiratory  coefficient  in,  71. 


Diabetes,  sugar   content   of   blood 
in,  68. 

tolerance  for  sugar  and  protein 
in,  68. 

relation  to  ductless  glands,  64. 
Diacetic  acid,  79. 

determination  of,  86. 

origin  of,  80. 

tests  for,  84. 
Digestion  1. 

mechanical  factors  in,  2,  13. 

products  of  gastric,  2,  9. 

products  of  intestinal,  12. 

products  of  salivary,  1. 

purpose  of,  2. 
Duodenal    juice,     examination    of, 
25. 

Ehrlich's  aldehyde  reaction,  91,  95. 
Ehrlich's  diazo  reaction,  91,  95. 
Enzymes,    amylopsin,    12,    25. 

enterokinase,  12. 

erepsin,  12. 

lactase,  12. 

lipase,  12,  26. 

maltase,  12. 

pepsin,  1,  2,  4,  7. 

pepsinogen,  2. 

ptyalin,  1. 

rennin,  1,  9. 

sucrase,  12. 

thrombin,  115. 

trypsin,  12,  26. 

trypsinogen,  12. 
Epithelial    cells,    in    urinary    sedi- 
ments, 99. 
Erythrocytes,  in  urinary  sediments, 

98. 
Exudates,  118,  124. 

Fat,  digestion  of,  12. 

in  feces,  17. 

in  milk,  103,  111. 
.      oxidation  of,  69.  79,  80,  81. 

utilization  of,  18. 
Feces,  12-25. 

albumin  in,  tests  for,  23. 

amount  of,  15. 


INDEX. 


133 


Feces,  bacteria  of,  14. 

blood  in,  tests  for,  23. 

carbohydrate  residues  of,  18. 

chemical  examination  of,  22,  23, 
24. 

color  of,  15,  20. 

connective  tissue  remains  in,  19, 
21. 

consistency  of,  15,  21. 

etheral  extract  of,  17. 

fat  in,  19. 

fermentation  test  in,  22. 

gall-stones  in,  24,  25. 

method  of  collection  of,  20. 

microchemical    examination    of, 
21,  22. 

mucus  in,  19. 

muscle  remains  in,  20,  21. 

nature  of,  15. 

nitrogenous  substances  in,  16. 

normal,  15. 

odor  of,  16,  20. 

products  of  intestinal   putrefac- 
tion in,  14. 

reaction  of,  16,  22. 
Formaldehyde,      detection     of     in 

milk,  113. 
Functional  tests,  phenolsulphoneph- 
thalein,  53,  58. 

galactose,  73. 

lactose,  58. 

levulose,  73.       \ 

potassium  iodide,  58. 

Schmidt-Strasburger,  18,  19. 

Schmidt's  nuclei,  20. 

sodium  chloride,  58. 

urea,  53. 

Galactose,  in  urine,  73. 
Gastric  contents,  1-11. 

acidity  of,  3,  5,  6,  7. 

blood  in,  4,  9. 

color  of,  5. 

consistency  of,  5. 

enzymes  of,  4,  7,  8,  9. 

lactic  acid  in,  3,  7. 

microscopical  examination  of,  11. 

mucus  in,  5. 


Gastric  contents,  odor  of,  3,  5. 

Salomon  test  on,  9. 

sediment  in,  5. 

tryptophane  test  on,  10. 

volume  of,  3,  5. 
Glucose,  conversion  of  to  fat,  61. 
63. 

conversion  to  glycogen,  60. 

influence    of    internal    secretions 
upon   metabolism   of,    64. 

in  urine,  60. 

quantitative     determination    of, 
76. 

oxidation  of,  60. 

test  for,  74. 
Glucosuria,  60-78. 
Glycogen,  in  liver,  60. 

in  muscle,  61. 
Glycogenesis,  60. 

defect  in,  62,  63,  68. 
Glycogenolysis,  60. 

excessive,  62,  63,  68. 
Glycosuria,  see  glucosuria. 

Hematoporphyrin,  in  urine,  89. 

test  for,  92. 
Hippuric  acid,  41. 
Hydrogen  peroxide,  detection  of  .in 

milk,  113. 
/j-hydroxybutyric  acid,  79. 

determination  of,  86. 

origin  of,  80. 

test  for,  85. 
Hyperglucemia,  63,  66,  68,  72. 
Hypoglucemia,  63,  66. 
Hypophysis,  65. 

Indican,  17,  90. 

quantitative    determination     of, 
93. 

test  for,  93. 
Indicators,  alizarin,  7. 

congo  red,  5,  44. 

dimethylaminoazobenzine,  5. 

ferric  ammonium  alum,  42. 

phenolphthalein,  6,  43. 
Indole,  16. 
Indoleacetic  acid  (urorosein),  91. 


134 


INDEX. 


Intestinal  juice,  enzymes  of,  12,  25. 
Intestines,    absorption  in,    13. 
peristaltic  movements  of,  13. 

Lactose,  in  milk,  105. 

in  urine,  58,  73,  78. 
Leucine,  52. 

in  urinary  sediments,  101. 
Leucocytes,  98. 
Levulose,  61. 

in  urine,  72,  78. 

Magnesium,  in  urine,  33. 
Maltose,  12,  74. 
Melanins,  in  urine,  90. 

detection  of,  93. 
Milk,  103-113. 

adaptation  of  in  nutrition,   105, 
106,  110. 

influence  of  food  upon,  109. 

mineral  matter  of,  105. 

modification  of,  107. 

organic  extractives  of,  105. 

pasteurization  of,  109. 

preservatives  in,  113. 

protein  of,  104,  112. 

qualitative  composition  of,  103. 

quantitative    relations    of,     105, 
106,  110. 

reaction  of,  103,  111. 

secretion  of,  108,  109. 

specific  gravity  of,  103,  110,  111. 

sterilization  of,  109. 

sugar  of,  105,  112. 

total  solids  of,  111. 
Mucous  cylinders,  97. 

Nephritis,  50,  51,  52,  53,  59,  63,  68. 
Nitrogen,    total,    determination   of 
in  urine,  44. 

non-protein,  in  blood,  120. 

non-protein,  in  milk,  112. 
Nucleoprotein,.  51. 

detection  of,  57. 

occurrence  of,  51. 

significance  of,  51. 

Oxalic  acid,  41,  100.    , 


Pancreas,  64. 

Pancreatic  juice,  action  of  secretin 
upon,  12. 

enzymes  of,  12,  25. 
Pentose,  72. 
Pentosuria,  72. 

Phenolsulphonephthalein  test,  58. 
Phosphates,  in  milk,  105. 

in  urine,  32,  43,  100. 
Pigmenturia,  88-95. 
Potassium,  in  urine,  33. 
Preservatives,  detection  of  in  milk, 
113. 

for  urine,  41. 
Protein,  digestion  of,  2,  12. 

in  blood,  115. 

in  milk,  104,  105,  112. 

in  urine,  49. 

requirement  of,  1. 

utilization  of  17. 
Proteose,  51. 

detection  of,  57. 
Pus  cells,  see  leucocytes. 

Qualitative  tests,  see  tests. 
Quantitative  determination  of, 
in  blood. 

glucose,  119. 

non-protein  nitrogen,  120. 
urea,  122. 
uric  acid,  122. 
in  duodenal  juice, 
amylopsin,  25. 
lipase,  26. 
trypsin,  26. 
in  gastric  contents. 

acidities,  free,  combined,  total, 

5.    ' 
lactic  acid,  7. 
pepsin,  8. 
in  milk. 

acidity,  111. 
fat,  111. 
lactose,  112. 
protein,  112. 
specific  gravity,  111. 
total  solids,  111. 


INDEX. 


135 


Quantitative  determination  of, 
in  transudates  and  exudates. 

protein,  124. 

specific  gravity,  124. 
in  urine. 

acetone,  86. 

albumin,  55,  56,  57. 

amino  acids,  58. 

ammonia,  43. 

chlorides,  42. 

creatine,  48. 

creatinine,  47. 

diacetic  acid,  86. 

glucose,  76,  77. 

/3-hydroxybutyric  acid,  86. 

indican,  93. 

phosphates,  43. 

renal  efficiency,  58. 

specific  gravity,  42. 

total  acidity,  43. 

total  nitrogen,  44. 

total  solids,  42. 

urea,  45.- 

uric  acid,  47. 

Reaction,  of  feces,  16,  22. 

of  food  materials  in  alimentary 
tract,  13. 

of  milk,  103,  111. 

of  urine,  30,  31,  41. 
Reagents,    qualitative    and  quanti- 
tative, preparation  of, 

accessory     solution,     for     phos- 
phates, 43. 

alizarin,  7. 

Almen's  reagent,  57. 

ammonium  sulphocyanate  stand- 
ard, 42. 

Arnold-Lipliawsky's   reagent,  85. 

Barfoed's  reagent,  76. 

Benedict's    qualitative    reagent, 
75. 

Benedict's  quantitative   reagent, 
77. 

Bial's  reagent,  78. 

bromine  water,  10. 

casein  solution,  26. 

congo  red,  5,  44. 


Reagents,      dimethylaminoazoben- 
zine,  5. 

Ehrlich's  aldehyde  reagent,  95. 
•    Ehrlich's  diazo  reagent,  95. 

Esbach's  reagent,  56. 

Fehling's  solution,  75. 

Folin-  Denis    standard  uric    acid 
solution,  124. 

Folin- Macallum     uric    acid    re- 
agent, 123. 

Folin-Shaffer  uric  acid  reagent, 47. 

gelatin,  for  trypsin  test,  27. 

guaiac  solution,  9. 

hypobromite  solution,  46. 

hydrochloric  acid,   approximate- 
ly normal,  48. 

hydrochloric  acid,   tenth-normal 
6. 

hydrochloric  acid,  0 .  6  per  cent. ,  8. 

indigo  blue  solution,  standard,  94. 

iodine  solution,  21. 

Nessler's  reagent,  121. 

neutral  formaldehyde,  43. 

Nylander's  reagent,  75. 

Obermayer's  reagent,  93. 

pea  globulin  solution,  8. 

phenolphthalein,  6. 

phenolphthalin,  24. 

phosphotungstic    acid    solution, 
112. 

potassium       bichromate,       half- 
normal,  47. 

potassium  permanganate,  twen- 
tieth-normal, 47. 

Robert's  reagent,  54. 

silver  nitrate,  standard,  42. 

Seliwanoff's  reagent,  78. 

sodium    hydroxide, tenth-normal, 
6,  44. 

sulphuric  acid,  tenth-normal,  6, 
44. 

Sudan  III,  21. 

Tsuchiya's  reagent,  56. 

Tdpfer's  reagent,  5. 

uranium  nitrate,  standard,  43. 

uric  acid  solution,  standard,  124. 
Records,  methods  of  keeping,  127- 
130. 


136 


INDEX. 


Reference  books,  130. 
Renal  disease,   metabolism  in,   52. 
Renal  efficiency,  estimation  of,  see^ 
functional  tests. 

Saccharose,  74. 
Secretin,  12. 
Secretion,  gastric,  1,  3. 

mammary,  108. 

pancreatic,  12. 

urinary,  28. 
Sediments,  urinary,  examination  of, 
96-102. 

organized  constituents  of,  96-99. 

unorganized  constituents  of,  96, 
99,  100,  101. 
Skatole,  in  feces,  16. 
Sodium,  in  urine,  33. 
Solutions,  see  reagents. 
Stomach,  absorption  in,  2. 

bacterial  action,  in  3. 

motility  of,  4. 

peristaltic  waves  of,  2. 

size  of,  2. 
Sulphates,  in  urine,  33. 
Sugars,  see  glucose,  etc. 

Tests  for,  qualitative, 
in  feces. 

albumin,  23. 

bile,  22. 

carbohydrate  remains,  21,  22. 

connective  tissue,  21. 

fat  and  fatty  acid  crystals,  21, 

gall  stones,  24. 

mucus,  21. 

muscle  fibers,  21. 

occult  blood,  23. 
in  gastric  contents. 

carcinoma  of  stomach,  9. 

blood,  9. 

free  hydrochloric  acid,  5. 

lactic  acid,  7. 

motility,  4. 

mucus,  5. 

pepsin,  7. 

rennin,  9. 


Tests  for,  qualitative, 
in  milk. 

boric  acid,  113. 

formaldehyde,  113. 

hydrogen  peroxide,  113. 
in  urine. 

acetone,  84.  . 

albumin,  54. 

bile  pigments,  92. 

diacetic  acid,  84. 

Ehrlich's     aldehyde     reaction, 
95. 

Ehrlich's  diazo  reaction,  95. 

galactose,  78. 

glucose,  74-76,  126. 

glucuronates,  78. 

hematoporphyrin,  92. 

p-hydroxybutyric  acid,  85. 

indican,  93. 

indoleacetic  acid  (urorosein) ,  95 

lactose,  78. 

levulose,  78. 

melanin,  93. 

nucleoprotein,  57. 

pentose,  78. 

proteoses,  57. 

urobilin,  92. 

urobilinogen,  95. 
Tests,      quantitative,     see      under 

quantitative    determinations. 
Test  meals,  Ewald-Boas,  3,  4. 
retention,  4. 

Schmidt-Strasburger,  18,  19. 
Thyroids,  64,  65. 
Transudates,  118,  124. 
Tyrosine,  52. 

in  urinary  sediments,  101. 

Urea,  in  blood,  116,  122. 

changes  in  elimination  of,  35,  36, 
52. 

determination  of,  45,  46,  122. 

origin  of,  35. 
Uremia,  59,  116. 
Uric  acid,  amount  of  in  urine,  37. 

changes  in  elimination  of,  37. 

determination  of,  47,  122. 

endogenous,  exogenous,  37. 


INDEX 


137 


Uric  acid,  fate  of,  37. 

in  blood,  116,  122-124. 

influence      of      atophan      upon 
elimination  of,  38. 

in  urinary  calculi,  100,  101. 

in  urinary  sediments,  100,  101. 

lithia  therapy,  38.    • 

origin  of,  36. 

piperazine  therapy,  38. 

relation  of  to  gout,  37. 
Urine,  28-102. 

acetone  in  79,  80,  84,  86. 

albumin  in,  49,  116. 

amino  acids  in,  49,  51,  52. 

ammonia  in,  34,  43,  82,  84. 

bile  in,  89,  92. 

blood  in,  90,  93,  98. 

calcium  in,  33. 

chlorides  in,  32,  42,  53,  58. 

color  of,  30,  42,  88. 

creatine  in,  40,  48. 

-creatinine  in,  38,  39,  47. 

detection  of,  in  other  fluids,  124. 

diacetic  acid  in,  79,  80,  84,  86. 

galactose  in,  73,  78. 

globulin  in,  49. 

glucose  in,  60,  74. 

hematoporphyrin  in,  89,  92. 

hippuric  acid  in,  41. 

p-hydroxybutyric    acid    in,    79, 
80,  85,  86. 


Urine,  indican  in,  17,  90,  93,  94. 

lactose  in,  73,  78. 

levulose  in,  62,  72,  78. 

magnesium  in,  33. 

melanin  in,  90". 

nucleoprotein  in,  51,  57. 

odor  of,  31. 

oxalic  acid  in,  41,  100. 

pentoses  in,  72,  78. 

pigments  in,  88. 

phosphates  in,  32. 

potassium  in,  33. 

preservation  of,  41,  102. 

proteoses  in,  51,  57. 

purines  in,  34,  36,  47. 

reaction  of,  30,  31,  41. 

sodium  in,  33. 

specific  gravity  of,  30,  42. 

sulphates  in,  33. 

total  solids  in,  42. 

total  nitrogen  in,  34,  44. 

transparency  of,  31. 

urea  in,  35,  45,  53,  58. 

uric  acid  in,  36,  47. 
,     volume  of,  28,  42. 
Urobilin,  88,  92. 
Urobilinogen,  88,  91,  95. 
Urochrome,  88. 
Uroerythrin,  89. 

Yeasts,  96,  99. 


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